P97-antibody conjugates and methods of use

ABSTRACT

The present invention provides p97-antibody conjugates and related compositions and methods, which may be used in any of a variety of therapeutic methods, including methods for the treatment of cancers such as Her2/neu-expressing and Her1/EGFR-expressing cancers.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.13/542,435, filed Jul. 5, 2012; which claims the benefit under 35 U.S.C.§119(e) of U.S. Application No. 61/658,217, filed Jun. 11, 2012, andU.S. Application No. 61/504,646, filed Jul. 5, 2011, each of which isincorporated by reference in its entirety.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is BIOA_(—)003_(—)03 US_ST25.txt. The text file isabout 41 KB, was created on Aug. 27, 2015, and is being submittedelectronically via EFS-Web.

BACKGROUND

1. Technical Field

The present invention relates generally to p97-antibody conjugates,related compositions and methods of using the same. Certain embodimentsare more specifically directed to conjugates comprising a p97polypeptide sequence and an antibody or antigen-binding fragment thereofthat specifically binds to a cell surface receptor or cell surfaceprotein, or a cancer-associated antigen, such as the human Her2/neuprotein or the Her1/EGF receptor. Such antibody conjugates are useful,for example, in methods for treating a variety of diseases, includingoncological diseases such as Her2/neu-expressing andHer1/EGFR-expressing cancers.

2. Description of the Related Art

Overcoming the difficulties of delivering therapeutic agents to specificregions of the brain represents a major challenge to treatment of mostbrain disorders. In its neuroprotective role, the blood-brain barrier(BBB) functions to hinder the delivery of many potentially importantdiagnostic and therapeutic agents to the brain. Therapeutic moleculesand genes that might otherwise be effective in diagnosis and therapy donot cross the BBB in adequate amounts. It is reported that over 95% ofall therapeutic molecules do not cross the blood-brain barrier.

Trastuzumab (tradename Herceptin®), an approved monoclonal antibodyspecific for the human Her2/neu protein, is an important therapeuticoption in the treatment of the approximately 30% of human breast cancersthat are positive for this protein. While trastuzumab has provenvaluable in the treatment and control of systemic disease; it cannotaddress the frequently observed spread of metastatic Her2/neu-expressingcancer cells in the central nervous system (CNS), due to the fact thattrastuzumab cannot cross the blood-brain barrier.

There is an important unmet need for improving the therapeutic potentialof antibodies, including those that are specific for Her2/neu orHer1/EGFR. For example, there is a need for anti-Her2/neu oranti-Her1/EGFR antibodies and antigen-binding fragments that haveimproved activity and/or other properties relative to conventionalantibodies. In addition, there is a need for compositions and methodsthat facilitate the delivery of anti-Her2/neu or anti-Her1/EGFRantibodies across the blood-brain-barrier in order to effectively treatHer2/neu+ or Her1/EGFR+ cancers, particularly those that havemetastasized to the CNS. These same needs apply to other cancerantigen-specific antibodies, including antibodies that are specific forHer3, A33 antigen, CD5, CD19, CD20, CD22, CD23 (IgE Receptor), C242antigen, 5T4, IL-6, IL-13, vascular endothelial growth factor VEGF(e.g., VEGF-A), and others.

The present invention addresses these needs and offers other relatedadvantages.

BRIEF SUMMARY

According to a general aspect, the present invention providestherapeutic compositions comprising a p97 polypeptide sequence and anantibody or an antigen-binding fragment thereof. In some embodiments,the antibody or fragment thereof specifically binds to a cell surfaceprotein, such as a cell surface receptor. In particular embodiments, theantibody specifically binds to a cancer-associated antigen, or cancerantigen. Certain cancer antigens include cell surface proteins and theirrespective ligands. In specific embodiments, the antibody or fragmentthereof specifically binds the human Her2/neu protein or the humanHer1/EGF receptor.

In certain aspects, the p97 polypeptide sequence and the antibody orfragment thereof are each bound to or encapsulated within a particle,e.g., a nanoparticle, bead, lipid formulation, lipid particle, orliposome, e.g., immunoliposome. In particular embodiments, the p97polypeptide sequence is present on the surface of the particle, and theantibody or fragment thereof is present on surface of the particleand/or encapsulated within the particle.

In a related general aspect, the present invention also providestherapeutic conjugates comprising a p97 polypeptide sequence covalentlylinked to an antibody or antigen-binding fragment thereof. In specificembodiments, the antibody or fragment thereof specifically binds thehuman Her2/neu protein. As described herein, such compositions andconjugates are of particular value in the treatment ofHer2/neu-expressing cancers, including those which have metastasized tothe CNS.

The p97 polypeptide sequence used in the conjugates of the invention canbe essentially any amino acid sequence derived from a p97 protein. In aspecific embodiment, the p97 polypeptide sequence used in a conjugate ofthe invention comprises the amino acid sequence set forth in SEQ IDNO: 1. In another specific embodiment, the p97 polypeptide sequence is asequence having at least 80% identity to the sequence of SEQ ID NO: 1.In still another specific embodiment, the p97 polypeptide sequence is afragment of a human p97 protein sequence having at least about 20, 30,40, 50, 60, 70, 80, 90 or 100, or more contiguous amino acid residues ofthe sequence set forth in SEQ ID NO: 1. In another specific embodiment,the p97 polypeptide sequence is a soluble p97 polypeptide sequence. Instill another specific embodiment, the p97 polypeptide sequence is asequence that is effective for facilitating transport of an antibody towhich it is linked across the blood-brain barrier.

A p97 polypeptide sequence can be conjugated to any therapeutic antibodyor antigen-binding fragment (e.g., anti-Her2/neu antibody orantigen-binding fragment) using any of a variety of known andestablished methodologies, illustrative examples of which are describedherein. These techniques include chemical conjugation techniques. Inother embodiments, the techniques rely upon standard recombinant DNAtechnology (e.g., for producing fusion polypeptides).

In certain more specific embodiments of the invention, the p97polypeptide sequence is covalently linked to the antibody orantigen-binding fragment with a linker. In a more specific embodiment,the p97 polypeptide sequence is (a) covalently linked to the antibody orantigen-binding fragment with a polymeric cross-linker, (b) covalentlylinked to the antibody or antigen-binding fragment via a nanoparticle,or (c) operatively linked to the antibody or antigen-binding fragmentthereof via a liposome. In another specific embodiment, the p97polypeptide sequence is covalently linked to the antibody orantigen-binding fragment with a polymeric cross-linker comprisingpolyethylene glycol. In another specific embodiment, the p97 polypeptidesequence is covalently linked to the antibody or antigen-bindingfragment with a polymeric cross-linker comprising thioether linkage(s).

The anti-Her2/neu antibody or antigen-binding fragment thereof used inaccordance with the invention will generally be capable of specificallybinding to a human Her2/neu protein having a sequence set forth in SEQID NO: 2.

In a more specific embodiment, the anti-Her2/neu antibody is trastuzumabor an antigen-binding fragment or derivative thereof.

The p97 antibody conjugate can also be a fusion polypeptide comprising ap97 polypeptide sequence and a Her2/neu specific antibody or antigenbinding sequence. The fusion polypeptides may be advantageouslyco-expressed using routine recombinant DNA methodologies to produce adesired conjugate of the invention.

Accordingly, in another aspect, the present invention provides isolatedfusion polynucleotides, and host cells containing the same, wherein thefusion polynucleotides encode fusion polypeptides comprising a p97polypeptide sequence and therapeutic antibody or antigen-bindingfragment thereof, for instance, a Her2/neu specific antibody or antigenbinding fragment.

According to still another aspect, the present invention providespharmaceutical compositions comprising a p97 antibody conjugate or apolynucleotide encoding a p97 conjugate, and a pharmaceuticallyacceptable excipient.

According to still another aspect, the invention provides a method forthe treatment of a subject with a Her2/neu-expressing cancer byadministering to the subject a pharmaceutical composition comprising ap97-antibody conjugate of the invention. The Her2/neu-expressing cancerto be treated is, in certain embodiments, a metastatic cancer,particularly a metastatic cancer characterized by CNS progression.

Also included are conjugates, comprising a p97 polypeptide sequencecovalently linked to a monoclonal antibody or antigen-binding fragmentthereof. In some embodiments, the antibody or antigen-binding fragmentthereof specifically binds to a eukaryotic cell-surface protein. Incertain embodiments, the antibody or antigen-binding fragment thereofspecifically binds to a mammalian cell-surface protein, optionally ahuman cell-surface protein. In particular embodiments, the antibody orantigen binding fragment thereof specifically binds to acancer-associated antigen.

In certain embodiments, the cancer-associated antigen is associated withone or more of breast cancer, metastatic brain cancer, prostate cancer,gastrointestinal cancer, lung cancer, ovarian cancer, testicular cancer,head and neck cancer, stomach cancer, bladder cancer, pancreatic cancer,liver cancer, kidney cancer, squamous cell carcinoma, CNS or braincancer, melanoma, non-melanoma cancer, thyroid cancer, endometrialcancer, epithelial tumor, bone cancer, or a hematopoietic cancer.

In some embodiments, the cancer-associated antigen is selected from oneor more of Her2/neu, Her1/EGF receptor (EGFR), Her3, A33 antigen, CD5,CD19, CD20, CD22, CD23 (IgE Receptor), C242 antigen, 5T4, IL-6, IL-13,vascular endothelial growth factor VEGF (e.g., VEGF-A) VEGFR-1, VEGFR-2,CD30, CD33, CD37, CD40, CD44, CD51, CD52, CD56, CD74, CD80, CD152,CD200, CD221, CCR4, HLA-DR, CTLA-4, NPC-1C, tenascin, vimentin,insulin-like growth factor 1 receptor (IGF-1R), alpha-fetoprotein,insulin-like growth factor 1 (IGF-1), carbonic anhydrase 9 (CA-IX),carcinoembryonic antigen (CEA), integrin α_(v)β₃, integrin α₅β₁, folatereceptor 1, transmembrane glycoprotein NMB, fibroblast activationprotein, alpha (FAP), glycoprotein 75, TAG-72, MUC1, MUC16 (or CA-125),phosphatidylserine, prostate-specific membrane antigen (PMSA), NR-LU-13antigen, TRAIL-R1, tumor necrosis factor receptor superfamily member 10b(TNFRSF10B or TRAIL-R2), SLAM family member 7 (SLAMF7), EGP40pancarcinoma antigen, B-cell activating factor (BAFF), platelet-derivedgrowth factor receptor, glycoprotein EpCAM (17-1A), Programmed Death-1,protein disulfide isomerase (PDI), Phosphatase of Regenerating Liver 3(PRL-3), prostatic acid phosphatase, Lewis-Y antigen, GD2 (adisialoganglioside expressed on tumors of neuroectodermal origin),glypican-3 (GPC3), and mesothelin.

In certain embodiments, the monoclonal antibody is selected from one ormore of trastuzumab, 3F8, abagovomab, adecatumumab, afutuzumab,alemtuzumab, alacizumab (pegol), amatuximab, apolizumab, bavituximab,bectumomab, belimumab, bevacizumab, bivatuzumab (mertansine),brentuximab vedotin, cantuzumab (mertansine), cantuzumab (ravtansine),capromab (pendetide), catumaxomab, cetuximab, citatuzumab (bogatox),cixutumumab, clivatuzumab (tetraxetan), conatumumab, dacetuzumab,dalotuzumab, detumomab, drozitumab, ecromeximab, edrecolomab,elotuzumab, enavatuzumab, ensituximab, epratuzumab, ertumaxomab,etaracizumab, farletuzumab, FBTA05, figitumumab, flanvotumab, galiximab,gemtuzumab, ganitumab, gemtuzumab (ozogamicin), girentuximab,glembatumumab (vedotin), ibritumomab tiuxetan, icrucumab, igovomab,indatuximab ravtansine, intetumumab, inotuzumab ozogamicin, ipilimumab(MDX-101), iratumumab, labetuzumab, lexatumumab, lintuzumab,lorvotuzumab (mertansine), lucatumumab, lumiliximab, mapatumumab,matuzumab, milatuzumab, mitumomab, mogamulizumab, moxetumomab(pasudotox), nacolomab (tafenatox), naptumomab (estafenatox),narnatumab, necitumumab, nimotuzumab, nivolumab, Neuradiab® (with orwithout radioactive iodine), NR-LU-10, ofatumumab, olaratumab,onartuzumab, oportuzumab (monatox), oregovomab, panitumumab, patritumab,pemtumomab, pertuzumab, pritumumab, racotumomab, radretumab,ramucirumab, rilotumumab, rituximab, robatumumab, samalizumab,sibrotuzumab, siltuximab, tabalumab, taplitumomab (paptox), tenatumomab,teprotumumab, TGN1412, ticilimumab, tremelimumab, tigatuzumab, TNX-650,tositumomab, TRBS07, tucotuzumab (celmoleukin), ublituximab, urelumab,veltuzumab, volociximab, votumumab, and zalutumumab, includingantigen-binding fragments thereof.

In specific embodiments, the monoclonal antibody is a humanized orchimeric monoclonal antibody.

Also included are pharmaceutical compositions comprising a p97-antibodyconjugate described herein and a pharmaceutically acceptable carrier orexcipient.

Certain embodiments relate to methods for the treatment of a subjectwith a cancer, comprising administering to the subject a pharmaceuticalcomposition described herein. In some embodiments, the subject has acancer selected from one or more of breast cancer, metastatic braincancer, prostate cancer, gastrointestinal cancer, lung cancer, ovariancancer, testicular cancer, head and neck cancer, stomach cancer, bladdercancer, pancreatic cancer, liver cancer, kidney cancer, squamous cellcarcinoma, CNS or brain cancer, melanoma, non-melanoma cancer, thyroidcancer, endometrial cancer, epithelial tumor, bone cancer, or ahematopoietic cancer.

In particular embodiments, the cancer is associated with expression ofat least one of Her2/neu, Her1/EGFR, Her3, A33 antigen, CD5, CD19, CD20,CD22, CD23 (IgE Receptor), C242 antigen, 5T4, IL-6, IL-13, vascularendothelial growth factor VEGF (e.g., VEGF-A) VEGFR-1, VEGFR-2, CD30,CD33, CD37, CD40, CD44, CD51, CD52, CD56, CD74, CD80, CD152, CD200,CD221, CCR4, HLA-DR, CTLA-4, NPC-1C, tenascin, vimentin, insulin-likegrowth factor 1 receptor (IGF-1R), alpha-fetoprotein, insulin-likegrowth factor 1 (IGF-1), carbonic anhydrase 9 (CA-IX), carcinoembryonicantigen (CEA), integrin α_(v)β₃, integrin α₅β₁, folate receptor 1,transmembrane glycoprotein NMB, fibroblast activation protein, alpha(FAP), glycoprotein 75, TAG-72, MUC1, MUC16 (or CA-125),phosphatidylserine, prostate-specific membrane antigen (PMSA), NR-LU-13antigen, TRAIL-R1, tumor necrosis factor receptor superfamily member 10b(TNFRSF10B or TRAIL-R2), SLAM family member 7 (SLAMF7), EGP40pancarcinoma antigen, B-cell activating factor (BAFF), platelet-derivedgrowth factor receptor, glycoprotein EpCAM (17-1A), Programmed Death-1,protein disulfide isomerase (PDI), Phosphatase of Regenerating Liver 3(PRL-3), prostatic acid phosphatase, Lewis-Y antigen, GD2 (adisialoganglioside expressed on tumors of neuroectodermal origin),glypican-3 (GPC3), or mesothelin. In certain embodiments, the monoclonalantibody portion of the p97-antibody conjugate specifically binds to thecancer-associated antigen.

In certain embodiments, the cancer is a metastatic colorectal cancer ora head and neck cancer, and the monoclonal antibody specifically bindsto Her1/EGFR and is an EGFR antagonist. In particular embodiments, themonoclonal antibody specifically binds to (e.g., one or more continuousor discontinuous epitopes of) SEQ ID NO:15 (Her1/EGFR). In certainembodiments, the cancer is an EGFR-expressing metastatic colorectalcancer. In specific embodiments, the colorectal cancer is KRASwild-type. In certain embodiments, the conjugate is administered afterfailure of both irinotecan- and oxiplatin-based regimens. In someembodiments, the subject is intolerant to irinotecan-based regimens oris refractory to irinotecan-based chemotherapy. In other aspects, thecancer is a locally or regionally advanced squamous cell carcinoma ofthe head and neck, a recurrent locoregional disease or metastaticsquamous cell carcinoma of the head and neck, or a recurrent ormetastatic squamous cell carcinoma of the head and neck progressingafter platinum-based therapy. In some embodiments, the conjugate isadministered in combination with radiation therapy, platinum-basedtherapy, or platinum-based therapy with 5-FU. In certain of these andrelated embodiments, the antibody is cetuximab, or an antigen-bindingfragment thereof.

Certain conjugates comprise a p97 polypeptide covalently linked to anantibody (Ab) according to one of the structures:

p97(FGly)-R₁-Ab or p97-R₁-(FGly)Ab

where R₁ is at least one aldehyde reactive linkage; and FGly is aformylglycine residue within a heterologous sulfatase motif thatcomprises the structure:

(SEQ ID NO: 5) X₁(FGly)X₂Z₂X₃

where Z₂ is a proline or alanine residue; X₁ is present or absent and,when present, is any amino acid, where X₁ is optionally present when theheterologous sulfatase motif is at the N-terminus of the p97polypeptide; and X₂ and X₃ are each independently any amino acid.

In some embodiments, R₁ comprises a Schiff base. In particularembodiments, R₁ is an oxime linkage, a hydrazine linkage, or a hydrazinecarbothiamide linkage.

Also included are isolated p97 polypeptides, comprising at least oneheterologous sulfatase motif that comprises the following structure:

(SEQ ID NO: 6) X₁Z₁X₂Z₂X₃

where Z₁ is cysteine or serine; Z₂ is a proline or alanine residue; X₁is present or absent and, when present, is any amino acid, where X₁ isoptionally present when the heterologous sulfatase motif is at theN-terminus of the aldehyde tagged polypeptide; and X₂ and X₃ are eachindependently any amino acid.

Certain isolated p97 polypeptides comprise at least one heterologoussulfatase motif that comprises the structure:

(SEQ ID NO: 5) X₁(FGly)X₂Z₂X₃

where FGly is a formylglycine residue; Z₂ is a proline or alanineresidue; X₁ is present or absent and, when present, is any amino acid,where X₁ is optionally present when the heterologous sulfatase motif isat the N-terminus of the p97 polypeptide; and X₂ and X₃ are eachindependently any amino acid.

In some embodiments, the isolated p97 polypeptide is covalently linkedto an antibody (Ab) that comprises at least one heterologous sulfatasemotif, where the motif comprises the structure:

(SEQ ID NO: 5) X₁(FGly)X₂Z₂X₃

where FGly is a formylglycine residue; Z₂ is a proline or alanineresidue; X₁ is present or absent and, when present, is any amino acid,where X₁ is optionally present when the heterologous sulfatase motif isat the N-terminus of the antibody; and X₂ and X₃ are each independentlyany amino acid, where the p97 polypeptide and the antibody arecovalently linked via their respective FGly residues to form ap97-antibody conjugate. In some embodiments, the isolated p97-antibodyconjugate comprises the following structure:

p97(FGly)-R₁-L-R₂-(FGly)Ab

where R₁ and R₂ are the same or different aldehyde reactive linkage; andL is a linker moiety.

In some embodiments, the at least one heterologous sulfatase motif is atthe C-terminus of the p97 polypeptide and the N-terminus of theantibody. In certain embodiments, the at least one heterologoussulfatase motif is at the N-terminus of the p97 polypeptide and theC-terminus of the antibody. In particular embodiments, the at least oneheterologous sulfatase motif is at the N-terminus of the p97 polypeptideand the N-terminus of the antibody. In some embodiments, the at leastone heterologous sulfatase motif is at the C-terminus of the p97polypeptide and the C-terminus of the antibody. In specific embodiments,R₁ and R₂ independently comprise a Schiff base. In certain instances, R₁and R₂ are independently an oxime linkage, a hydrazide linkage, or ahydrazine carbothiamide linkage. In some instances, L is a peptide, awater-soluble polymer, a detectable label, or a glycan.

Also included are methods of producing a p97 polypeptide, comprising a)culturing a host cell that expresses an introduced polynucleotide, wherethe introduced polynucleotide encodes the p97 polypeptide of claim 20,and where the host cell expresses a formylglycine generating enzyme(FGE) which converts Z₁ into a formylglycine (FGly) residue; and b)isolating the 97 polypeptide from the cell. In some embodiments, the p97polypeptide comprises (i) at least one unnatural amino acid with anazide side-chain, or (ii) at least one unnatural amino acid with analkyne side-chain.

Certain embodiments relate to conjugate, comprising the structure (I) or(II):

where R is a p97 polypeptide and R¹ is an antibody or antigen-bindingfragment thereof; or where R is an antibody or antigen-binding fragmentthereof and R¹ is a p97 polypeptide. In some embodiments, the antibodyspecifically binds the human Her2/neu protein, or other cell surfaceprotein or cancer associated antigen described herein. Particularexamples include Her1/EGF receptor (EGFR), Her3, A33 antigen, CD5, CD19,CD20, CD22, CD23 (IgE Receptor), C242 antigen, 5T4, IL-6, IL-13,vascular endothelial growth factor VEGF (e.g., VEGF-A) VEGFR-1, VEGFR-2,CD30, CD33, CD37, CD40, CD44, CD51, CD52, CD56, CD74, CD80, CD152,CD200, CD221, CCR4, HLA-DR, CTLA-4, NPC-1C, tenascin, vimentin,insulin-like growth factor 1 receptor (IGF-1R), alpha-fetoprotein,insulin-like growth factor 1 (IGF-1), carbonic anhydrase 9 (CA-IX),carcinoembryonic antigen (CEA), integrin α_(v)β₃, integrin α₅β₁, folatereceptor 1, transmembrane glycoprotein NMB, fibroblast activationprotein, alpha (FAP), glycoprotein 75, TAG-72, MUC1, MUC16 (or CA-125),phosphatidylserine, prostate-specific membrane antigen (PMSA), NR-LU-13antigen, TRAIL-R1, tumor necrosis factor receptor superfamily member 10b(TNFRSF10B or TRAIL-R2), SLAM family member 7 (SLAMF7), EGP40pancarcinoma antigen, B-cell activating factor (BAFF), platelet-derivedgrowth factor receptor, glycoprotein EpCAM (17-1A), Programmed Death-1,protein disulfide isomerase (PDI), Phosphatase of Regenerating Liver 3(PRL-3), prostatic acid phosphatase, Lewis-Y antigen, GD2 (adisialoganglioside expressed on tumors of neuroectodermal origin),glypican-3 (GPC3), and mesothelin.

In specific embodiments, the antibody is trastuzumab, 3F8, abagovomab,adecatumumab, afutuzumab, alemtuzumab, alacizumab (pegol), amatuximab,apolizumab, bavituximab, bectumomab, belimumab, bevacizumab, bivatuzumab(mertansine), brentuximab vedotin, cantuzumab (mertansine), cantuzumab(ravtansine), capromab (pendetide), catumaxomab, cetuximab, citatuzumab(bogatox), cixutumumab, clivatuzumab (tetraxetan), conatumumab,dacetuzumab, dalotuzumab, detumomab, drozitumab, ecromeximab,edrecolomab, elotuzumab, enavatuzumab, ensituximab, epratuzumab,ertumaxomab, etaracizumab, farletuzumab, FBTA05, figitumumab,flanvotumab, galiximab, gemtuzumab, ganitumab, gemtuzumab (ozogamicin),girentuximab, glembatumumab (vedotin), ibritumomab tiuxetan, icrucumab,igovomab, indatuximab ravtansine, intetumumab, inotuzumab ozogamicin,ipilimumab (MDX-101), iratumumab, labetuzumab, lexatumumab, lintuzumab,lorvotuzumab (mertansine), lucatumumab, lumiliximab, mapatumumab,matuzumab, milatuzumab, mitumomab, mogamulizumab, moxetumomab(pasudotox), nacolomab (tafenatox), naptumomab (estafenatox),narnatumab, necitumumab, nimotuzumab, nivolumab, NEURADIAB® (with orwithout radioactive iodine), NR-LU-10, ofatumumab, olaratumab,onartuzumab, oportuzumab (monatox), oregovomab, panitumumab, patritumab,pemtumomab, pertuzumab, pritumumab, racotumomab, radretumab,ramucirumab, rilotumumab, rituximab, robatumumab, samalizumab,sibrotuzumab, siltuximab, tabalumab, taplitumomab (paptox), tenatumomab,teprotumumab, TGN1412, ticilimumab, tremelimumab, tigatuzumab, TNX-650,tositumomab, TRBS07, tucotuzumab (celmoleukin), ublituximab, urelumab,veltuzumab, volociximab, votumumab, or zalutumumab, or anantigen-binding fragment thereof.

Also included are methods of producing a p97-antibody conjugate,comprising: (a) performing an azide-alkyne cycloaddition reactionbetween: (i) a p97 polypeptide that comprises at least one unnaturalamino acid with an azide side-chain and an antibody or antigen-bindingfragment thereof that comprises at least one unnatural amino acid withan alkyne side-chain; or (ii) a p97 polypeptide that comprises at leastone unnatural amino acid with an alkyne side-chain and an antibody orantigen-binding fragment thereof that comprises at least one unnaturalamino acid with an azide side-chain; and (b) isolating a p97-antibodyconjugate from the reaction, thereby producing a p97-antibody conjugate.

These and other aspects of the present invention will become apparentupon reference to the following detailed description and attacheddrawings. All references disclosed herein are hereby incorporated byreference in their entirety as if each was incorporated individually.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D show the results of cell viability assays for the humanbreast cancer cell line BT474.

FIGS. 2A-2D show the results of cell viability assays for the humanbreast cancer cell line MCF7-HER2.

FIGS. 3A-3D show the results of cell viability assays for the humanbreast cancer cell line MCF7-vector.

FIGS. 4A-4D show the results of cell viability assays for the humanbreast cancer cell line SKBR3.

FIGS. 5A-5D show the biodistribution of rhodamine(rhod)-labeled proteinsin the brain tissue of mice. In these figures, “MTF” is p97, “BTA” istrastuzumab, and “MTF-BTA” is a p97-trastuzumab conjugate.

FIGS. 6A-6F show the distribution of ¹²⁵I-labeled trastuzumab in themouse brain at 24 hours post-intravenous administration. FIG. 6A showsbrain metastases of heterogenous size within the regions outlined inred, and FIG. 6B shows Texas Red-Dextran staining of the metastases.FIG. 6C shows an autoradiogram of ¹²⁵I-labeled trastuzumab, andindicates the fold increase in antibody relative to the surroundingnormal brain tissue. As shown in FIG. 6F, the K_(in) values fortrastuzumab alone are about 1.46×10⁻⁷ mL/sec/g in normal brain tissueand about 3.8×10⁻⁷ mL/sec/g in brain metastases.

FIGS. 7A-7E show the distribution of ¹²⁵I-labeled trastuzumab in themouse brain and other tissues at 24 hours post-intravenousadministration. FIG. 7A shows brain metastases of heterogenous sizewithin the regions outlined in red, and FIG. 7B shows Texas Red-Dextranstaining of the metastases. FIG. 7C shows the autoradiogram of¹²⁵I-labeled trastuzumab, and indicates the fold increase in antibodyrelative to the surrounding normal brain tissue. FIG. 7D shows thetissue to blood ratio of ¹²⁵I-labeled trastuzumab in various tissues,and FIG. 7E shows the distribution in normal brain tissue and brainmetastases (Mets).

FIGS. 8A-8F show the distribution of ¹²⁵I-labeled p97-trastuzumab in themouse brain and other tissues at two hours post-intravenousadministration. FIG. 8A shows brain metastases of heterogenous sizewithin the regions outlined in red, and FIG. 8B shows Texas Red-Dextranstaining of the metastases. FIG. 8C shows an autoradiogram of¹²⁵I-labeled p97-trastuzumab conjugates, and the left of FIG. 8Cindicates the amount (ng/g) of conjugate found in each metastases. Theleft of FIG. 8B shows the fold increase of p97-trastuzumab conjugatefound in each metastases, relative to the brain distant to tumor (BDT)region shown in FIG. 8A. FIG. 8D shows the tissue/blood ratio ofp97-trastuzumab conjugate for a variety of tissues. FIG. 8E shows theratio of p97-trastuzumab conjugate in normal brain/blood and brainmetastases/blood. FIG. 8F summarizes the concentration of ¹²⁵I-labeledp97-trastuzumab conjugate found in individual brain metastases.

FIGS. 9A-9F show the distribution of ¹²⁵I-labeled p97-trastuzumab in themouse brain and other tissues at eight hours post-intravenousadministration. FIG. 9A shows brain metastases of heterogenous sizewithin the regions outlined in red, and FIG. 9B shows Texas Red-Dextranstaining of the metastases. FIG. 9C shows an autoradiogram of¹²⁵I-labeled p97-trastuzumab conjugate, and the left of FIG. 9Cindicates the amount (ng/g) of conjugate found in each metastases. Theleft of FIG. 9B shows the fold increase of p97-trastuzumab conjugatefound in each metastases, relative to the brain distant to tumor (BDT)regions shown in FIG. 9A. FIG. 9D shows the tissue/blood ratio ofp97-trastuzumab conjugate for a variety of tissues. FIG. 9E shows theratio of p97-trastuzumab conjugate in normal brain/blood and brainmetastases/blood. FIG. 9F summarizes the concentration of ¹²⁵I-labeledp97-trastuzumab conjugate found in individual brain metastases.

FIGS. 10A-10E summarize the data from the two and eight hour time pointsfollowing intravenous administration of ¹²⁵I-labeled p97-trastuzumabconjugate.

FIG. 10A shows the tissue/blood ratio of p97-trastuzumab conjugate for avariety of tissues. FIG. 10B shows that the levels of conjugate innormal brain tissue are marginally higher at the eight hour time point(relative to the two hour time point), and the levels of conjugate inbrain metastases are significantly higher at the that same time point.FIG. 10C shows the measured K_(in) values for the p97-trastuzumabconjugate in normal brain tissue (1.1×10⁴ mL/sec/g) and brain metastases(4.9×10⁴ mL/sec/g). FIG. 10D shows the percentage of injected dose inbrain tissue at 2 and 8 hours, and FIG. 10E summarizes the concentrationof ¹²⁵I-labeled p97-trastuzumab conjugate in individual brain metastasesat two and eight hours post-administration.

FIGS. 11A and 11B show HPLC analysis of p97-cetuximab conjugates. FIG.11A shows the HPLC profile of the crude reaction mixture after 24 hoursat room temperature, and FIG. 11B shows the size-exclusion HPLC profileof purified 1:1 p97-cetuximab conjugate (>96% purity, HPLC detection at220 nm).

FIG. 12 shows an SDS-PAGE analysis of the purified p97-cetuximabconjugate relative to the crude reaction mixture and p97 and cetuximabalone.

FIG. 13 is an Assay Plate Layout that shows the dilutions of Test andControl Articles (see Example 2).

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is the amino acid sequence of the human p97melanotransferrin protein (NP_(—)005920.2).

SEQ ID NO:2 is the amino acid sequence of the human Her2/neu protein(NP_(—)004439.2).

SEQ ID NO:3 is a nucleic acid sequence encoding the polypeptide sequenceof SEQ ID NO: 1.

SEQ ID NO:4 is a nucleic acid sequence of the encoding the polypeptidesequence of SEQ ID NO: 2.

SEQ ID NOS:5 and 6 are peptide sulfatase motifs.

SEQ ID NOS:8-14 are peptide linkers.

SEQ ID NO:15 is the amino acid sequence of the human Her1/epidermalgrowth factor receptor (EGFR).

DETAILED DESCRIPTION

The present invention relates generally to conjugate moleculescomprising p97 polypeptide sequences linked to antibodies orantigen-binding fragments thereof. Also included are compositions thatcomprise a p97 polypeptide sequence and an antibody or antigen-bindingfragment thereof, such as particle-based compositions, e.g., liposomes.The p97 polypeptide sequences of the invention can be conjugated to orcomposed with any antibody or antigen-binding fragment thereof,including therapeutic and diagnostic antibodies. Certain therapeuticand/or diagnostic antibodies or antigen-binding fragments thereofspecifically bind to a cell surface protein, such as a cell surfacereceptor.

In particular embodiments, the antibodies or fragments specifically bindto the human Her2/neu protein. As demonstrated herein, trastuzumab(trade name Herceptin®), a humanized monoclonal antibody used clinicallyin the treatment of HER2+ breast cancer, was chemically linked to p97polypeptide sequences to generate p97-antibody conjugates. Unexpectedly,the p97-antibody conjugates demonstrated a significant improvement incancer killing activity compared to trastuzumab alone. Furthermore, theresults confirmed, as expected, that trastuzumab does not enter humanbrain endothelial (HBE) cells in culture. However, in the case ofp97-antibody conjugates, there was a marked transport of the conjugatesinto HBE cells, indicating that the conjugates have the potential toenter brain tissue. The combination of p97 and trastuzumab as a proteinconjugate also synergistically increased delivery across the blood brainbarrier to parenchymal brain tissue, relative to delivery of p97 aloneand trastuzumab alone. Based on these unexpected findings, the presentinvention provides compositions and methods for the improved treatmentof Her-2/neu-expressing cancers, including those associated withmetastasis to the CNS. The present invention further providesp97-antibody conjugates, compositions, and related methods for theimproved treatment of other types of cancer, particularly those thatassociate with at least one antigen that can be targeted by antibodytherapy.

The practice of the present invention will employ, unless indicatedspecifically to the contrary, conventional methods of virology,immunology, microbiology, molecular biology and recombinant DNAtechniques within the skill of the art, many of which are describedbelow for the purpose of illustration. Such techniques are explainedfully in the literature. See, e.g., Current Protocols in MolecularBiology or Current Protocols in Immunology, John Wiley & Sons, New York,N.Y. (2009); Ausubel et al., Short Protocols in Molecular Biology,3^(rd) ed., Wiley & Sons, 1995; Sambrook and Russell, Molecular Cloning:A Laboratory Manual (3rd Edition, 2001); Maniatis et al. MolecularCloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach,vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed.,1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985);Transcription and Translation (B. Hames & S. Higgins, eds., 1984);Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guideto Molecular Cloning (1984) and other like references.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural references unless the contentclearly dictates otherwise.

Throughout this specification, unless the context requires otherwise,the word “comprise,” or variations such as “comprises” or “comprising,”will be understood to imply the inclusion of a stated element or integeror group of elements or integers but not the exclusion of any otherelement or integer or group of elements or integers.

Each embodiment in this specification is to be applied mutatis mutandisto every other embodiment unless expressly stated otherwise.

Standard techniques may be used for recombinant DNA, oligonucleotidesynthesis, and tissue culture and transformation (e.g., electroporation,lipofection). Enzymatic reactions and purification techniques may beperformed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. These and relatedtechniques and procedures may be generally performed according toconventional methods well known in the art and as described in variousgeneral and more specific references that are cited and discussedthroughout the present specification. Unless specific definitions areprovided, the nomenclature utilized in connection with, and thelaboratory procedures and techniques of, molecular biology, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques may be used for recombinant technology,molecular biological, microbiological, chemical syntheses, chemicalanalyses, pharmaceutical preparation, formulation, and delivery, andtreatment of patients.

The functional properties of the p97-antibody conjugates describedherein may be assessed using a variety of methods known to the skilledperson, including, e.g., affinity/binding assays (for example, surfaceplasmon resonance, competitive inhibition assays); cytotoxicity assays,cell viability assays, cell proliferation or differentiation assays,cancer cell and/or tumor growth inhibition using in vitro or in vivomodels. Other assays may test the ability of conjugates described hereinto block normal Her2/neu-mediated responses. The conjugates describedherein may also be tested for effects on receptor internalisation, invitro and in vivo efficacy, etc. Such assays may be performed usingwell-established protocols known to the skilled person (see e.g.,Current Protocols in Molecular Biology (Greene Publ. Assoc. Inc. & JohnWiley & Sons, Inc., NY, NY); Current Protocols in Immunology (Edited by:John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach,Warren Strober 2001 John Wiley & Sons, NY, NY); or commerciallyavailable kits.

P97 Polypeptide Sequences

As noted above, exemplary conjugate molecules and compositions of thepresent invention include a p97 polypeptide sequence. The term“polypeptide” is used in its conventional meaning, i.e., as a sequenceof amino acids. The polypeptides are not limited to a specific length ofthe product; thus, peptides, oligopeptides, and proteins are includedwithin the definition of polypeptide, and such terms may be usedinterchangeably herein unless specifically indicated otherwise. Thisterm also does not refer to or exclude post-expression modifications ofthe polypeptide, for example, glycosylations, acetylations,phosphorylations and the like, as well as other modifications known inthe art, both naturally occurring and non-naturally occurring. Apolypeptide may be an entire protein, or a subsequence thereof.

In certain specific embodiments, a p97 polypeptide sequence used in aconjugate of the invention comprises the human p97 sequence set forth inSEQ ID NO: 1.

In other specific embodiments, a p97 polypeptide sequence used in aconjugate of the invention comprises a sequence having at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity, along itslength, to a human p97 sequence set forth in SEQ ID NO: 1.

In still other specific embodiments, a p97 polypeptide sequence used ina conjugate of the invention comprises a fragment of a human p97sequence set forth in SEQ ID NO: 1, e.g., wherein the fragment comprisesat least about 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100, or more,contiguous amino acids, including all intermediate lengths, of a humanp97 sequence set forth in SEQ ID NO: 1.

In other specific embodiments, a p97 polypeptide sequence used in aconjugate of the invention comprises a fragment of a human p97 sequenceset forth in SEQ ID NO: 1, wherein the fragment consists of no more thanabout 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100, or more, contiguousamino acids, including all intermediate lengths, of a human p97 sequenceset forth in SEQ ID NO: 1.

In still other specific embodiments, a p97 polypeptide sequence used ina conjugate of the invention comprises a fragment of a human p97sequence set forth in SEQ ID NO: 1, wherein the fragment includes about20-500, 20-400, 20-300, 20-200, 20-100, or 20-50, contiguous amino acidsof a human p97 sequence set forth in SEQ ID NO: 1.

In certain other embodiments, p97 polypeptide sequences of interest areamino acid subsequences and variants of p97 that are effective fortransporting an anti-Her2/neu antibody across the blood brain barrier.

In other specific embodiments, a p97 polypeptide sequence used in aconjugate is a soluble form of a p97 polypeptide (e.g., Yang et al.,Prot Exp Purif. 34:28-48, 2004), or a fragment or variant thereof. Insome aspects, the p97 polypeptide has a deletion of the all or a portionof the hydrophobic domain (residues 710-738 of SEQ ID NO:1), alone or incombination with a deletion of all or a portion of the signal peptide(residues 1-19 of SEQ ID NO:1). In specific aspects, the p97 polypeptidecomprises or consists of residues 20-711 of SEQ ID NO:1, includingvariants and fragments thereof.

In certain other embodiments, the p97 fragment or variant used in aconjugate of the invention is a fragment or variant capable of binding ap97 receptor, a LRP1 receptor and/or a LRP1B receptor.

It will be understood that a conjugate may also comprise additionalamino acids unrelated to the p97 and anti-Her2/neu antibody sequencespresent.

The p97 polypeptide sequence may also be a variant p97 polypeptidesequence. A p97 polypeptide “variant,” as the term is used herein, is apolypeptide that typically differs from a p97 polypeptide specificallydisclosed herein in one or more substitutions, deletions, additionsand/or insertions. Such variants may be naturally occurring or may besynthetically generated, for example, by modifying one or more of theabove polypeptide sequences of the invention and evaluating theiractivity as described herein and/or using any of a number of techniqueswell known in the art.

In many instances, a variant will contain conservative substitutions. A“conservative substitution” is one in which an amino acid is substitutedfor another amino acid that has similar properties, such that oneskilled in the art of peptide chemistry would expect the secondarystructure and hydropathic nature of the polypeptide to be substantiallyunchanged. As described above, modifications may be made in thestructure of the polynucleotides and polypeptides of the presentinvention and still obtain a functional molecule that encodes a variantor derivative polypeptide with desirable characteristics. When it isdesired to alter the amino acid sequence of a polypeptide to create anequivalent, or even an improved, variant or portion of a polypeptide ofthe invention, one skilled in the art will typically change one or moreof the codons of the encoding DNA sequence according to Table 1.

For example, certain amino acids may be substituted for other aminoacids in a protein structure without appreciable loss of interactivebinding capacity with structures such as, for example, antigen-bindingregions of antibodies or binding sites on substrate molecules. Since itis the interactive capacity and nature of a protein that defines thatprotein's biological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence, and, of course, itsunderlying DNA coding sequence, and nevertheless obtain a protein withlike properties. It is thus contemplated that various changes may bemade in the peptide sequences of the disclosed compositions, orcorresponding DNA sequences which encode said peptides withoutappreciable loss of their utility.

TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys CUGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGAAGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr TACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte & Doolittle, 1982, incorporated herein byreference). It is accepted that the relative hydropathic character ofthe amino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like. Each amino acid has been assigned a hydropathicindex on the basis of its hydrophobicity and charge characteristics(Kyte & Doolittle, 1982). These values are: isoleucine (+4.5); valine(+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e., still obtaina biological functionally equivalent protein. In making such changes,the substitution of amino acids whose hydropathic indices are within ±2is preferred, those within ±1 are particularly preferred, and thosewithin ±0.5 are even more particularly preferred. It is also understoodin the art that the substitution of like amino acids can be madeeffectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101(specifically incorporated herein by reference in its entirety), statesthat the greatest local average hydrophilicity of a protein, as governedby the hydrophilicity of its adjacent amino acids, correlates with abiological property of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (O); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). It isunderstood that an amino acid can be substituted for another having asimilar hydrophilicity value and still obtain a biologically equivalent,and in particular, an immunologically equivalent protein. In suchchanges, the substitution of amino acids whose hydrophilicity values arewithin ±2 is preferred, those within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions that take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

Amino acid substitutions may further be made on the basis of similarityin polarity, charge, solubility, hydrophobicity, hydrophilicity and/orthe amphipathic nature of the residues. For example, negatively chargedamino acids include aspartic acid and glutamic acid; positively chargedamino acids include lysine and arginine; and amino acids with unchargedpolar head groups having similar hydrophilicity values include leucine,isoleucine and valine; glycine and alanine; asparagine and glutamine;and serine, threonine, phenylalanine and tyrosine. Other groups of aminoacids that may represent conservative changes include: (1) ala, pro,gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile,leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. Avariant may also, or alternatively, contain nonconservative changes. Ina preferred embodiment, variant polypeptides differ from a nativesequence by substitution, deletion or addition of five amino acids orfewer. Variants may also (or alternatively) be modified by, for example,the deletion or addition of amino acids that have minimal influence onthe immunogenicity, secondary structure and hydropathic nature of thepolypeptide.

As noted above, polypeptides may comprise a signal (or leader) sequenceat the N-terminal end of the protein, which co-translationally orpost-translationally directs transfer of the protein. The polypeptidemay also be conjugated to a linker or other sequence for ease ofsynthesis, purification or identification of the polypeptide (e.g.,poly-His), or to enhance binding of the polypeptide to a solid support.For example, a polypeptide may be conjugated to an immunoglobulin Fcregion.

Polypeptides of the invention may be prepared using any of a variety ofwell known synthetic and/or recombinant techniques, the latter of whichare further described below. Polypeptides, portions and other variantsgenerally less than about 150 amino acids can be generated by syntheticmeans, using techniques well known to those of ordinary skill in theart. In one illustrative example, such polypeptides are synthesizedusing any of the commercially available solid-phase techniques, such asthe Merrifield solid-phase synthesis method, where amino acids aresequentially added to a growing amino acid chain. See Merrifield, J. Am.Chem. Soc. 85:2149-46 (1963). Equipment for automated synthesis ofpolypeptides is commercially available from suppliers such as PerkinElmer/Applied BioSystems Division (Foster City, Calif.), and may beoperated according to the manufacturer's instructions.

In general, polypeptide compositions (including fusion polypeptides) ofthe invention are isolated. An “isolated” polypeptide is one that isremoved from its original environment. For example, anaturally-occurring protein or polypeptide is isolated if it isseparated from some or all of the coexisting materials in the naturalsystem. Preferably, such polypeptides are also purified, e.g., are atleast about 90% pure, more preferably at least about 95% pure and mostpreferably at least about 99% pure.

When comparing polypeptide or polynucleotide sequences, two sequencesare said to be “identical” if the nucleotide or amino acid sequence inthe two sequences is the same when aligned for maximum correspondence,as described below. Comparisons between two sequences are typicallyperformed by comparing the sequences over a comparison window toidentify and compare local regions of sequence similarity. A “comparisonwindow” as used herein, refers to a segment of at least about 20contiguous positions, usually 30 to about 75, 40 to about 50, in which asequence may be compared to a reference sequence of the same number ofcontiguous positions after the two sequences are optimally aligned.

Optimal alignment of sequences for comparison may be conducted using theMegalign program in the Lasergene suite of bioinformatics software(DNASTAR, Inc., Madison, Wis.), using default parameters. This programembodies several alignment schemes described in the followingreferences: Dayhoff, M. O., A model of evolutionary change inproteins—Matrices for detecting distant relationships (1978). In Atlasof Protein Sequence and Structure, vol. 5, supp. 3, pp. 345-58 (Dayhoff,M. O., ed.); Hein J., Methods in Enzymology 183:626-45 (1990); Higginset al., CABIOS 5:151-53 (1989); Myers et al., CABIOS 4:11-17 (1988);Robinson, E. D., Comb. Theor 11:105 (1971); Saitou et al., Mol. Biol.Evol. 4:406-25 (1987); Sneath et al., Numerical Taxonomy—the Principlesand Practice of Numerical Taxonomy (1973); Wilbur et al., Proc. Natl.Acad. Sci. USA 80:726-30 (1983).

Alternatively, optimal alignment of sequences for comparison may beconducted by the local identity algorithm of Smith et al., Add. APL.Math 2:482 (1981), by the identity alignment algorithm of Needleman etal., J. Mol. Biol. 48:443 (1970), by the search for similarity methodsof Pearson et al., Proc. Natl. Acad. Sci. USA 85:2444 (1988), bycomputerized implementations of these algorithms (GAP, BESTFIT, BLAST,FASTA, and TFASTA in the Wisconsin Genetics Software Package, GeneticsComputer Group (GCG), 575 Science Dr., Madison, Wis.), or by inspection.

One preferred example of algorithms that are suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nucl.Acids Res. 25:3389-3402 (1977), and Altschul et al., J. Mol. Biol.215:403-10 (1990), respectively. BLAST and BLAST 2.0 can be used, forexample with the parameters described herein, to determine percentsequence identity for the polynucleotides and polypeptides of theinvention. Software for performing BLAST analyses is publicly availablethrough the National Center for Biotechnology Information. For aminoacid sequences, a scoring matrix can be used to calculate the cumulativescore. Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, Tand X determine the sensitivity and speed of the alignment.

In one preferred approach, the “percentage of sequence identity” isdetermined by comparing two optimally aligned sequences over a window ofcomparison of at least 20 positions, wherein the portion of thepolypeptide or polynucleotide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) of 20 percent or less,usually 5 to 15 percent, or 10 to 12 percent, as compared to thereference sequences (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical amino acid ornucleic acid residue occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the reference sequence (i.e., the window size)and multiplying the results by 100 to yield the percentage of sequenceidentity.

Antibodies

The antibody or antigen-binding fragment used in the conjugates orcompositions of the present invention can be of essentially any type.Particular examples include therapeutic and diagnostic antibodies. As iswell known in the art, an antibody is an immunoglobulin molecule capableof specific binding to a target, such as a carbohydrate, polynucleotide,lipid, polypeptide, etc., through at least one epitope recognition site,located in the variable region of the immunoglobulin molecule. As usedherein, the term encompasses not only intact polyclonal or monoclonalantibodies, but also fragments thereof (such as dAb, Fab, Fab′, F(ab′)₂,Fv), single chain (ScFv), synthetic variants thereof, naturallyoccurring variants, fusion proteins comprising an antibody portion withan antigen-binding fragment of the required specificity, humanizedantibodies, chimeric antibodies, and any other modified configuration ofthe immunoglobulin molecule that comprises an antigen-binding site orfragment (epitope recognition site) of the required specificity.“Diabodies,” multivalent or multispecific fragments constructed by genefusion (WO94/13804; P. Holliger et al., Proc. Natl. Acad. Sci. USA 906444-6448, 1993) are also a particular form of antibody contemplatedherein. Minibodies comprising a scFv joined to a CH3 domain are alsoincluded herein (S. Hu et al., Cancer Res., 56, 3055-3061, 1996). Seee.g., Ward, E. S. et al., Nature 341, 544-546 (1989); Bird et al.,Science, 242, 423-426, 1988; Huston et al., PNAS USA, 85, 5879-5883,1988); PCT/US92/09965; WO94/13804; P. Holliger et al., Proc. Natl. Acad.Sci. USA 90 6444-6448, 1993; Y. Reiter et al., Nature Biotech, 14,1239-1245, 1996; S. Hu et al., Cancer Res., 56, 3055-3061, 1996.

The term “antigen-binding fragment” as used herein refers to apolypeptide fragment that contains at least one CDR of an immunoglobulinheavy and/or light chains that binds to the antigen of interest, forinstance, the human Her2/neu protein. In this regard, an antigen-bindingfragment of the herein described antibodies may comprise 1, 2, 3, 4, 5,or all 6 CDRs of a VH and VL sequence from antibodies that bind to atherapeutic or diagnostic target, such as human Her2/neu.

The term “antigen” refers to a molecule or a portion of a moleculecapable of being bound by a selective binding agent, such as anantibody, and additionally capable of being used in an animal to produceantibodies capable of binding to an epitope of that antigen. An antigenmay have one or more epitopes.

The term “epitope” includes any determinant, preferably a polypeptidedeterminant, capable of specific binding to an immunoglobulin or T-cellreceptor. An epitope is a region of an antigen that is bound by anantibody. In certain embodiments, epitope determinants includechemically active surface groupings of molecules such as amino acids,sugar side chains, phosphoryl or sulfonyl, and may in certainembodiments have specific three-dimensional structural characteristics,and/or specific charge characteristics. In certain embodiments, anantibody is said to specifically bind an antigen when it preferentiallyrecognizes its target antigen in a complex mixture of proteins and/ormacromolecules. An antibody is said to specifically bind an antigen whenthe equilibrium dissociation constant is 10⁻⁷ or 10⁻⁸ M. In someembodiments, the equilibrium dissociation constant may be 10⁻⁹ M or10⁻¹⁰ M.

In certain embodiments, antibodies and antigen-binding fragments thereofas described herein include a heavy chain and a light chain CDR set,respectively interposed between a heavy chain and a light chainframework region (FR) set which provide support to the CDRs and definethe spatial relationship of the CDRs relative to each other. As usedherein, the term “CDR set” refers to the three hypervariable regions ofa heavy or light chain V region. Proceeding from the N-terminus of aheavy or light chain, these regions are denoted as “CDR1,” “CDR2,” and“CDR3” respectively. An antigen-binding site, therefore, includes sixCDRs, comprising the CDR set from each of a heavy and a light chain Vregion. A polypeptide comprising a single CDR, (e.g., a CDR1, CDR2 orCDR3) is referred to herein as a “molecular recognition unit.”Crystallographic analysis of a number of antigen-antibody complexes hasdemonstrated that the amino acid residues of CDRs form extensive contactwith bound antigen, wherein the most extensive antigen contact is withthe heavy chain CDR3. Thus, the molecular recognition units areprimarily responsible for the specificity of an antigen-binding site.

As used herein, the term “FR set” refers to the four flanking amino acidsequences which frame the CDRs of a CDR set of a heavy or light chain Vregion. Some FR residues may contact bound antigen; however, FRs areprimarily responsible for folding the V region into the antigen-bindingsite, particularly the FR residues directly adjacent to the CDRs. WithinFRs, certain amino residues and certain structural features are veryhighly conserved. In this regard, all V region sequences contain aninternal disulfide loop of around 90 amino acid residues. When the Vregions fold into a binding-site, the CDRs are displayed as projectingloop motifs which form an antigen-binding surface. It is generallyrecognized that there are conserved structural regions of FRs whichinfluence the folded shape of the CDR loops into certain “canonical”structures—regardless of the precise CDR amino acid sequence. Further,certain FR residues are known to participate in non-covalent interdomaincontacts which stabilize the interaction of the antibody heavy and lightchains.

The structures and locations of immunoglobulin variable domains may bedetermined by reference to Kabat, E. A. et al., Sequences of Proteins ofImmunological Interest. 4th Edition. US Department of Health and HumanServices. 1987, and updates thereof, now available on the Internet(immuno.bme.nwu.edu).

A “monoclonal antibody” refers to a homogeneous antibody populationwherein the monoclonal antibody is comprised of amino acids (naturallyoccurring and non-naturally occurring) that are involved in theselective binding of an epitope. Monoclonal antibodies are highlyspecific, being directed against a single epitope. The term “monoclonalantibody” encompasses not only intact monoclonal antibodies andfull-length monoclonal antibodies, but also fragments thereof (such asFab, Fab′, F(ab′)₂, Fv), single chain (ScFv), variants thereof, fusionproteins comprising an antigen-binding portion, humanized monoclonalantibodies, chimeric monoclonal antibodies, and any other modifiedconfiguration of the immunoglobulin molecule that comprises anantigen-binding fragment (epitope recognition site) of the requiredspecificity and the ability to bind to an epitope. It is not intended tobe limited as regards the source of the antibody or the manner in whichit is made (e.g., by hybridoma, phage selection, recombinant expression,transgenic animals, etc.). The term includes whole immunoglobulins aswell as the fragments etc. described above under the definition of“antibody”.

The proteolytic enzyme papain preferentially cleaves IgG molecules toyield several fragments, two of which (the F(ab) fragments) eachcomprise a covalent heterodimer that includes an intact antigen-bindingsite. The enzyme pepsin is able to cleave IgG molecules to provideseveral fragments, including the F(ab′)₂ fragment which comprises bothantigen-binding sites. An Fv fragment for use according to certainembodiments of the present invention can be produced by preferentialproteolytic cleavage of an IgM, and on rare occasions of an IgG or IgAimmunoglobulin molecule. Fv fragments are, however, more commonlyderived using recombinant techniques known in the art. The Fv fragmentincludes a non-covalent V_(H)::V_(L) heterodimer including anantigen-binding site which retains much of the antigen recognition andbinding capabilities of the native antibody molecule. Inbar et al.(1972) Proc. Nat. Acad. Sci. USA 69:2659-2662; Hochman et al. (1976)Biochem 15:2706-2710; and Ehrlich et al. (1980) Biochem 19:4091-4096.

In certain embodiments, single chain Fv or scFV antibodies arecontemplated. For example, Kappa bodies (III et al., Prot. Eng. 10:949-57 (1997); minibodies (Martin et al., EMBO J 13: 5305-9 (1994);diabodies (Holliger et al., PNAS 90: 6444-8 (1993); or Janusins(Traunecker et al., EMBO J 10: 3655-59 (1991) and Traunecker et al.,Int. J. Cancer Suppl. 7: 51-52 (1992), may be prepared using standardmolecular biology techniques following the teachings of the presentapplication with regard to selecting antibodies having the desiredspecificity.

A single chain Fv (sFv) polypeptide is a covalently linked V_(H)::V_(L)heterodimer which is expressed from a gene fusion including V_(H)- andV_(L)-encoding genes linked by a peptide-encoding linker. Huston et al.(1988) Proc. Nat. Acad. Sci. USA 85(16):5879-5883. A number of methodshave been described to discern chemical structures for converting thenaturally aggregated—but chemically separated—light and heavypolypeptide chains from an antibody V region into an sFv molecule whichwill fold into a three dimensional structure substantially similar tothe structure of an antigen-binding site. See, e.g., U.S. Pat. Nos.5,091,513 and 5,132,405, to Huston et al.; and U.S. Pat. No. 4,946,778,to Ladner et al.

In certain embodiments, an antibody as described herein is in the formof a diabody. Diabodies are multimers of polypeptides, each polypeptidecomprising a first domain comprising a binding region of animmunoglobulin light chain and a second domain comprising a bindingregion of an immunoglobulin heavy chain, the two domains being linked(e.g. by a peptide linker) but unable to associate with each other toform an antigen binding site: antigen binding sites are formed by theassociation of the first domain of one polypeptide within the multimerwith the second domain of another polypeptide within the multimer(WO94/13804). A dAb fragment of an antibody consists of a VH domain(Ward, E. S. et al., Nature 341, 544-546 (1989)).

Where bispecific antibodies are to be used, these may be conventionalbispecific antibodies, which can be manufactured in a variety of ways(Holliger, P. and Winter G. Current Opinion Biotechnol. 4, 446-449(1993)), e.g. prepared chemically or from hybrid hybridomas, or may beany of the bispecific antibody fragments mentioned above. Diabodies andscFv can be constructed without an Fc region, using only variabledomains, potentially reducing the effects of anti-idiotypic reaction.

Bispecific diabodies, as opposed to bispecific whole antibodies, mayalso be particularly useful because they can be readily constructed andexpressed in E. coli. Diabodies (and many other polypeptides such asantibody fragments) of appropriate binding specificities can be readilyselected using phage display (WO94/13804) from libraries. If one arm ofthe diabody is to be kept constant, for instance, with a specificitydirected against antigen X, then a library can be made where the otherarm is varied and an antibody of appropriate specificity selected.Bispecific whole antibodies may be made by knobs-into-holes engineering(J. B. B. Ridgeway et al., Protein Eng., 9, 616-621, 1996).

In certain embodiments, the antibodies described herein may be providedin the form of a UniBody®. A UniBody® is an IgG4 antibody with the hingeregion removed (see GenMab Utrecht, The Netherlands; see also, e.g.,US20090226421). This antibody technology creates a stable, smallerantibody format with an anticipated longer therapeutic window thancurrent small antibody formats. IgG4 antibodies are considered inert andthus do not interact with the immune system. Fully human IgG4 antibodiesmay be modified by eliminating the hinge region of the antibody toobtain half-molecule fragments having distinct stability propertiesrelative to the corresponding intact IgG4 (GenMab, Utrecht). Halving theIgG4 molecule leaves only one area on the UniBody® that can bind tocognate antigens (e.g., disease targets) and the UniBody® thereforebinds univalently to only one site on target cells. For certain cancercell surface antigens, this univalent binding may not stimulate thecancer cells to grow as may be seen using bivalent antibodies having thesame antigen specificity, and hence UniBody® technology may affordtreatment options for some types of cancer that may be refractory totreatment with conventional antibodies. The small size of the UniBody®can be a great benefit when treating some forms of cancer, allowing forbetter distribution of the molecule over larger solid tumors andpotentially increasing efficacy.

In certain embodiments, the antibodies of the present disclosure maytake the form of a nanobody. Nanobodies are encoded by single genes andare efficiently produced in almost all prokaryotic and eukaryotic hostse.g. E. coli (see e.g. U.S. Pat. No. 6,765,087), moulds (for exampleAspergillus or Trichoderma) and yeast (for example Saccharomyces,Kluyvermyces, Hansenula or Pichia (see e.g. U.S. Pat. No. 6,838,254).The production process is scalable and multi-kilogram quantities ofnanobodies have been produced. Nanobodies may be formulated as aready-to-use solution having a long shelf life. The Nanoclone method(see, e.g., WO 06/079372) is a proprietary method for generatingNanobodies against a desired target, based on automated high-throughputselection of B-cells.

In certain embodiments, the antibodies or antigen-binding fragmentsthereof are humanized. This refers to a chimeric molecule, generallyprepared using recombinant techniques, having an antigen-binding sitederived from an immunoglobulin from a non-human species and theremaining immunoglobulin structure of the molecule based upon thestructure and/or sequence of a human immunoglobulin. The antigen-bindingsite may comprise either complete variable domains fused onto constantdomains or only the CDRs grafted onto appropriate framework regions inthe variable domains. Epitope binding sites may be wild type or modifiedby one or more amino acid substitutions. This eliminates the constantregion as an immunogen in human individuals, but the possibility of animmune response to the foreign variable region remains (LoBuglio, A. F.et al., (1989) Proc Natl Acad Sci USA 86:4220-4224; Queen et al., PNAS(1988) 86:10029-10033; Riechmann et al., Nature (1988) 332:323-327).Illustrative methods for humanization of antibodies include the methodsdescribed in U.S. Pat. No. 7,462,697.

Another approach focuses not only on providing human-derived constantregions, but modifying the variable regions as well so as to reshapethem as closely as possible to human form. It is known that the variableregions of both heavy and light chains contain threecomplementarity-determining regions (CDRs) which vary in response to theepitopes in question and determine binding capability, flanked by fourframework regions (FRs) which are relatively conserved in a givenspecies and which putatively provide a scaffolding for the CDRs. Whennonhuman antibodies are prepared with respect to a particular epitope,the variable regions can be “reshaped” or “humanized” by grafting CDRsderived from nonhuman antibody on the FRs present in the human antibodyto be modified. Application of this approach to various antibodies hasbeen reported by Sato, K., et al., (1993) Cancer Res 53:851-856.Riechmann, L., et al., (1988) Nature 332:323-327; Verhoeyen, M., et al.,(1988) Science 239:1534-1536; Kettleborough, C. A., et al., (1991)Protein Engineering 4:773-3783; Maeda, H., et al., (1991) HumanAntibodies Hybridoma 2:124-134; Gorman, S. D., et al., (1991) Proc NatlAcad Sci USA 88:4181-4185; Tempest, P. R., et al., (1991) Bio/Technology9:266-271; Co, M. S., et al., (1991) Proc Natl Acad Sci USA88:2869-2873; Carter, P., et al., (1992) Proc Natl Acad Sci USA89:4285-4289; and Co, M. S. et al., (1992) J Immunol 148:1149-1154. Insome embodiments, humanized antibodies preserve all CDR sequences (forexample, a humanized mouse antibody which contains all six CDRs from themouse antibodies). In other embodiments, humanized antibodies have oneor more CDRs (one, two, three, four, five, six) which are altered withrespect to the original antibody, which are also termed one or more CDRs“derived from” one or more CDRs from the original antibody.

In certain embodiments, the antibodies of the present invention may bechimeric antibodies. In this regard, a chimeric antibody is comprised ofan antigen-binding fragment of an antibody operably linked or otherwisefused to a heterologous Fc portion of a different antibody. In certainembodiments, the heterologous Fc domain is of human origin. In otherembodiments, the heterologous Fc domain may be from a different Ig classfrom the parent antibody, including IgA (including subclasses IgA1 andIgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4),and IgM. In further embodiments, the heterologous Fc domain may becomprised of CH2 and CH3 domains from one or more of the different Igclasses. As noted above with regard to humanized antibodies, theantigen-binding fragment of a chimeric antibody may comprise only one ormore of the CDRs of the antibodies described herein (e.g., 1, 2, 3, 4,5, or 6 CDRs of the antibodies described herein), or may comprise anentire variable domain (VL, VH or both).

An epitope that “specifically binds” or “preferentially binds” (usedinterchangeably herein) to an antibody or a polypeptide is a term wellunderstood in the art, and methods to determine such specific orpreferential binding are also well known in the art.

A molecule is said to exhibit “specific binding” or “preferentialbinding” if it reacts or associates more frequently, more rapidly, withgreater duration and/or with greater affinity with a particular cell orsubstance than it does with alternative cells or substances. An antibody“specifically binds” or “preferentially binds” to a target if it bindswith greater affinity, avidity, more readily, and/or with greaterduration than it binds to other substances. For example, an antibodythat specifically or preferentially binds to a specific epitope is anantibody that binds that specific epitope with greater affinity,avidity, more readily, and/or with greater duration than it binds toother epitopes. It is also understood by reading this definition that,for example, an antibody (or moiety or epitope) that specifically orpreferentially binds to a first target may or may not specifically orpreferentially bind to a second target. As such, “specific binding” or“preferential binding” does not necessarily require (although it caninclude) exclusive binding. Generally, but not necessarily, reference tobinding means preferential binding.

Immunological binding generally refers to the non-covalent interactionsof the type which occur between an immunoglobulin molecule and anantigen for which the immunoglobulin is specific, for example by way ofillustration and not limitation, as a result of electrostatic, ionic,hydrophilic and/or hydrophobic attractions or repulsion, steric forces,hydrogen bonding, van der Waals forces, and other interactions. Thestrength, or affinity of immunological binding interactions can beexpressed in terms of the dissociation constant (K_(d)) of theinteraction, wherein a smaller K_(d) represents a greater affinity.Immunological binding properties of selected polypeptides can bequantified using methods well known in the art. One such method entailsmeasuring the rates of antigen-binding site/antigen complex formationand dissociation, wherein those rates depend on the concentrations ofthe complex partners, the affinity of the interaction, and on geometricparameters that equally influence the rate in both directions. Thus,both the “on rate constant” (K_(on)) and the “off rate constant”(K_(off)) can be determined by calculation of the concentrations and theactual rates of association and dissociation. The ratio ofK_(off)/K_(on) enables cancellation of all parameters not related toaffinity, and is thus equal to the dissociation constant K_(d). See,generally, Davies et al. (1990) Annual Rev. Biochem. 59:439-473.

In certain embodiments, the antibody or antigen-binding fragment thereofspecifically binds to a cancer-associated antigen, or cancer antigen.Exemplary cancer antigens include cell surface proteins such as cellsurface receptors. Also included as cancer-associated antigens areligands that bind to such cell surface proteins or receptors. Inspecific embodiments, the antibody or antigen-binding fragmentspecifically binds to a intracellular cancer antigen.

In some embodiments, the cancer that associates with the cancer antigenis one or more of breast cancer, metastatic brain cancer, prostatecancer, gastrointestinal cancer, lung cancer, ovarian cancer, testicularcancer, head and neck cancer, stomach cancer, bladder cancer, pancreaticcancer, liver cancer, kidney cancer, squamous cell carcinoma, CNS orbrain cancer, melanoma, non-melanoma cancer, thyroid cancer, endometrialcancer, epithelial tumor, bone cancer, or a hematopoietic cancer.

In particular embodiments, the antibody or antigen-binding fragmentspecifically binds to at least one cancer antigen selected from humanHer2/neu, Her1/EGF receptor, Her3, A33 antigen, CD5, CD19, CD20, CD22,CD23 (IgE Receptor), C242 antigen, 5T4, IL-6, IL-13, vascularendothelial growth factor VEGF (e.g., VEGF-A) VEGFR-1, VEGFR-2, CD30,CD33, CD37, CD40, CD44, CD51, CD52, CD56, CD74, CD80, CD152, CD200,CD221, CCR4, HLA-DR, CTLA-4, NPC-1C, tenascin, vimentin, insulin-likegrowth factor 1 receptor (IGF-1R), alpha-fetoprotein, insulin-likegrowth factor 1 (IGF-1), carbonic anhydrase 9 (CA-IX), carcinoembryonicantigen (CEA), integrin α_(v)β₃, integrin α₅β₁, folate receptor 1,transmembrane glycoprotein NMB, fibroblast activation protein alpha(FAP), glycoprotein 75, TAG-72, MUC1, MUC16 (or CA-125),phosphatidylserine, prostate-specific membrane antigen (PMSA), NR-LU-13antigen, TRAIL-R1, tumor necrosis factor receptor superfamily member 10b(TNFRSF10B or TRAIL-R2), SLAM family member 7 (SLAMF7), EGP40pancarcinoma antigen, B-cell activating factor (BAFF), platelet-derivedgrowth factor receptor, glycoprotein EpCAM (17-1A), Programmed Death-1,protein disulfide isomerase (PDI), Phosphatase of Regenerating Liver 3(PRL-3), prostatic acid phosphatase, Lewis-Y antigen, GD2 (adisialoganglioside expressed on tumors of neuroectodermal origin),glypican-3 (GPC3), and mesothelin.

In particular embodiments, the antibody or antigen-binding fragmentthereof specifically binds to the human Her2/neu protein. Essentiallyany anti-Her2/neu antibody, antigen-binding fragment or otherHer2/neu-specific binding agent may be used in producing thep97-antibody conjugates of the present invention. Illustrativeanti-Her2/neu antibodies are described, for example, in U.S. Pat. Nos.5,677,171; 5,720,937; 5,720,954; 5,725,856; 5,770,195; 5,772,997;6,165,464; 6,387,371; and 6,399,063, the contents of which areincorporated by reference in their entireties.

In some embodiments, the antibody or antigen-binding fragment thereofspecifically binds to the human Her1/EGFR (epidermal growth factorreceptor). Essentially any anti-Her1/EGFR antibody, antigen-bindingfragment or other Her1-EGFR-specific binding agent may be used inproducing the p97-antibody conjugates of the present invention.Illustrative anti-Her1/EGFR antibodies are described, for example, inU.S. Pat. Nos. 5,844,093; 7,132,511; 7,247,301; 7,595,378; 7,723,484;7,939,072; and 7,960,516, the contents of which are incorporated byreference in their entireties.

In certain embodiments, the antibody is an anti-cancer therapeuticantibody, including exemplary antibodies such as 3F8, abagovomab,adecatumumab, afutuzumab, alemtuzumab, alacizumab (pegol), amatuximab,apolizumab, bavituximab, bectumomab, belimumab, bevacizumab, bivatuzumab(mertansine), brentuximab vedotin, cantuzumab (mertansine), cantuzumab(ravtansine), capromab (pendetide), catumaxomab, cetuximab, citatuzumab(bogatox), cixutumumab, clivatuzumab (tetraxetan), conatumumab,dacetuzumab, dalotuzumab, detumomab, drozitumab, ecromeximab,edrecolomab, elotuzumab, enavatuzumab, ensituximab, epratuzumab,ertumaxomab, etaracizumab, farletuzumab, FBTA05, figitumumab,flanvotumab, galiximab, gemtuzumab, ganitumab, gemtuzumab (ozogamicin),girentuximab, glembatumumab (vedotin), ibritumomab tiuxetan, icrucumab,igovomab, indatuximab ravtansine, intetumumab, inotuzumab ozogamicin,ipilimumab (MDX-101), iratumumab, labetuzumab, lexatumumab, lintuzumab,lorvotuzumab (mertansine), lucatumumab, lumiliximab, mapatumumab,matuzumab, milatuzumab, mitumomab, mogamulizumab, moxetumomab(pasudotox), nacolomab (tafenatox), naptumomab (estafenatox),narnatumab, necitumumab, nimotuzumab, nivolumab, Neuradiab® (with orwithout radioactive iodine), NR-LU-10, ofatumumab, olaratumab,onartuzumab, oportuzumab (monatox), oregovomab, panitumumab, patritumab,pemtumomab, pertuzumab, pritumumab, racotumomab, radretumab,ramucirumab, rilotumumab, rituximab, robatumumab, samalizumab,sibrotuzumab, siltuximab, tabalumab, taplitumomab (paptox), tenatumomab,teprotumumab, TGN1412, ticilimumab, tremelimumab, tigatuzumab, TNX-650,tositumomab, TRBS07, trastuzumab, tucotuzumab (celmoleukin),ublituximab, urelumab, veltuzumab, volociximab, votumumab, andzalutumumab. Also included are fragments, variants, and derivatives ofthese antibodies

In specific embodiments, the anti-Her2/neu antibody used in a conjugateof the invention is trastuzumab (Herceptin®), or a fragment orderivative thereof. Trastuzumab is a Her2/neu-specific monoclonalantibody approved for the treatment of human breast cancer. Asdemonstrated herein, conjugation of p97 amino acid sequences to thetrastuzumab antibody unexpectedly resulted in greater levels of cancercell killing than with the trastuzumab antibody alone. Furthermore,there was a marked transport of the p97-antibody conjugates into HBEcells, suggesting that the conjugates will be effective for deliveryacross the blood-brain barrier. Further data strongly suggest thattherapeutically effective concentrations of p97-trastuzumab conjugatecan be achieved in brain tissue metastases, even by systemic (e.g.,intravenous) administration of such conjugates. These data also suggestthat p97 and trastuzumab work synergistically together to selectivelytarget p97-trastuzumab conjugates to brain metastases relative to normalbrain tissue, and at a significantly greater rate (1000 fold) thantrastuzumab alone. Conjugation to p97 thus not only increases transportof trastuzumab across the blood-brain barrier, but also the blood-tumorbarrier. Further, because of the reduced distribution to heart tissuesrelative to other tissues, these data suggest that conjugation to p97might reduce the cardiotoxic effects of antibodies such as trastuzumab.Accordingly, the p97-antibody conjugates of the invention, in certainembodiments, will give rise to particular advantages in the treatment ofHer2/neu-expressing cancers, including but not limited to those thathave metastasized to the CNS.

In some embodiments, an anti-Her2/neu binding fragment comprises one ormore of the CDRs of a Her2/neu antibody. In this regard, it has beenshown in some cases that the transfer of only the VHCDR3 of an antibodycan be performed while still retaining desired specific binding (Barbaset al., PNAS (1995) 92: 2529-2533). See also, McLane et al., PNAS (1995)92:5214-5218, Barbas et al., J. Am. Chem. Soc. (1994) 116:2161-2162.

In specific embodiments, the anti-Her1/EGFR antibody used in a conjugateof the invention is cetuximab (Erbitux®), or a fragment or derivativethereof. In certain embodiments, an anti-Her1/EGFR binding fragmentcomprises one or more of the CDRs of a Her1/EGFR antibody such ascetuximab. Cetuximab is approved for the treatment of head and neckcancer, and colorectal cancer. Cetuximab is composed of the Fv(variable; antigen-binding) regions of the 225 murine EGFR monoclonalantibody specific for the N-terminal portion of human EGFR with humanIgG1 heavy and kappa light chain constant (framework) regions.

Antibodies may be prepared by any of a variety of techniques known tothose of ordinary skill in the art. See, e.g., Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988.Monoclonal antibodies specific for a polypeptide of interest may beprepared, for example, using the technique of Kohler and Milstein, Eur.J. Immunol. 6:511-519, 1976, and improvements thereto. Also included aremethods that utilize transgenic animals such as mice to express humanantibodies. See, e.g., Neuberger et al., Nature Biotechnology 14:826,1996; Lonberg et al., Handbook of Experimental Pharmacology 113:49-101,1994; and Lonberg et al., Internal Review of Immunology 13:65-93, 1995.Particular examples include the VELOCIMMUNE® platform by REGENEREX®(see, e.g., U.S. Pat. No. 6,596,541).

Antibodies can also be generated or identified by the use of phagedisplay or yeast display libraries (see, e.g., U.S. Pat. No. 7,244,592;Chao et al., Nature Protocols. 1:755-768, 2006). Non-limiting examplesof available libraries include cloned or synthetic libraries, such asthe Human Combinatorial Antibody Library (HuCAL), in which thestructural diversity of the human antibody repertoire is represented byseven heavy chain and seven light chain variable region genes. Thecombination of these genes gives rise to 49 frameworks in the masterlibrary. By superimposing highly variable genetic cassettes(CDRs=complementarity determining regions) on these frameworks, the vasthuman antibody repertoire can be reproduced. Also included are humanlibraries designed with human-donor-sourced fragments encoding alight-chain variable region, a heavy-chain CDR-3, synthetic DNA encodingdiversity in heavy-chain CDR-1, and synthetic DNA encoding diversity inheavy-chain CDR-2. Other libraries suitable for use will be apparent topersons skilled in the art. The p97 polypeptides described herein andknown in the art may be used in the purification process in, forexample, an affinity chromatography step.

These just-described techniques are, in and of themselves, known as suchin the art. The skilled person will, however, be able to use suchtechniques to obtain antibodies or antigen-binding fragments thereof andto covalently couple them with p97 polypeptide sequences according toseveral embodiments of the invention described herein.

P97-Antibody Conjugates

Conjugation of a p97 polypeptide sequence and an antibody or bindingfragment thereof can be carried out using standard chemical, biochemicaland/or molecular techniques. Indeed, it will be apparent how to make ap97-antibody conjugate in light of the present disclosure usingavailable art-recognized methodologies. Of course, it will generally bepreferred when linking the primary components of a conjugate of thepresent invention that the conjugation techniques employed and theresulting linking chemistries do not substantially disturb the desiredfunctionality or activity of the individual components of the conjugate.

In certain embodiments, conjugates of the invention may employ anysuitable linking groups or types known in the art for joining aheterologous polypeptide sequence to an antibody or antigen-bindingsequence. Preferably, the antigen (e.g., human Her-2/neu protein, humanHer1/EGFR) binding ability and/or activity of conjugate is notsubstantially reduced as a result of the conjugation technique employed,for example, as compared to the unconjugated antibody, such as theunconjugated anti-Her2/neu antibody.

In certain embodiments, a p97 polypeptide sequence may be coupled (e.g.,covalently linked) to a suitable antibody or binding fragment thereof(e.g., anti-Her2/neu monoclonal antibody) either directly or indirectly(e.g., via a linker group). A direct reaction between a p97 polypeptidesequence and an antibody is possible when each possesses a substituentcapable of reacting with the other. For example, a nucleophilic group,such as an amino or sulfhydryl group, on one may be capable of reactingwith a carbonyl-containing group, such as an anhydride or an acidhalide, or with an alkyl group containing a good leaving group (e.g., ahalide) on the other.

Alternatively, it may be desirable to couple a p97 polypeptide sequenceand an antibody or binding fragment thereof (e.g., anti-Her2/neuantibody or binding fragment thereof) via a linker group. A linker groupcan also function as a spacer to distance an antibody from the p97polypeptide sequence in order to avoid interference with bindingcapabilities, targeting capabilities or other functionalities. A linkergroup can also serve to increase the chemical reactivity of asubstituent on an agent or an antibody, and thus increase the couplingefficiency. An increase in chemical reactivity may also facilitate theuse of agents, or functional groups on agents, which otherwise would notbe possible.

In some embodiments, it may be desirable to couple more than one p97polypeptide sequence to an antibody (e.g., anti-Her2/neu antibody), orvice versa. For example, in certain embodiments, multiple p97polypeptide sequences are coupled to one antibody molecule or bindingfragment thereof. In one embodiment, multiple p97 polypeptide sequencesare coupled to one anti-Her2/neu antibody molecule or binding fragmentthereof. The p97 polypeptide sequences can be the same or different.Regardless of the particular embodiment, conjugates containing multiplep97 polypeptide sequences may be prepared in a variety of ways. Forexample, more than one polypeptide may be coupled directly to anantibody molecule, or linkers that provide multiple sites for attachmentcan be used. Any of a variety of known heterobifunctional crosslinkingstrategies can be employed for making conjugates of the invention. Itwill be understood that many of these embodiments can be achieved bycontrolling the stoichiometries of the materials used during theconjugation/crosslinking procedure.

In a more specific embodiment of the invention, an amine-to-sulfhydrylcrosslinker is used for preparing a conjugate. In one preferredembodiment, for example, the crosslinker issuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC)(Thermo Scientific), which is a sulfhydryl crosslinker containingNHS-ester and maleimide reactive groups at opposite ends of amedium-length cyclohexane-stabilized spacer arm (8.3 angstroms). SMCC isa non-cleavable and membrane permeable crosslinker that can be used tocreate sulfhydryl-reactive, maleimide-activated anti-Her2/neu antibodiesor antigen-binding fragments for subsequent reaction with p97polypeptide sequences. NHS esters react with primary amines at pH 7-9 toform stable amide bonds. Maleimides react with sulfhydryl groups at pH6.5-7.5 to form stable thioether bonds. Thus, the amine reactive NHSester of SMCC crosslinks rapidly with primary amines of an anti-Her2/neuantibody and the resulting sulfhydryl-reactive maleimide group is thenavailable to react with cysteine residues of p97 to yield specificconjugates of interest.

In certain specific embodiments, the p97 polypeptide sequence ismodified to contain exposed sulfhydryl groups to facilitatecrosslinking, e.g., to facilitate crosslinking to a maleimide-activatedantibody, such as an anti-Her2/neu antibody. In a more specificembodiment, the p97 polypeptide sequence is modified with a reagentwhich modifies primary amines to add protected thiol sulfhydryl groups.In an even more specific embodiment, the reagentN-succinimidyl-S-acetylthioacetate (SATA) (Thermo Scientific) is used toproduce thiolated p97 polypeptides.

In other specific embodiments, a maleimide-activated antibody is reactedunder suitable conditions with thiolated p97 polypeptides to produce aconjugate of the present invention. It will be understood that bymanipulating the ratios of SMCC, SATA, antibody (e.g., anti-Her2/neuantibody) and p97 polypeptide in these reactions it is possible toproduce conjugates having differing stoichiometries, molecular weightsand properties.

The specific crosslinking strategy discussed above (and exemplifiedbelow) is but one of many examples of suitable conjugation strategiesthat may be employed in producing conjugates of the invention. It willbe evident to those skilled in the art that a variety of otherbifunctional or polyfunctional reagents, both homo- andhetero-functional (such as those described in the catalog of the PierceChemical Co., Rockford, Ill.), may be employed as the linker group.Coupling may be effected, for example, through amino groups, carboxylgroups, sulfhydryl groups or oxidized carbohydrate residues. There arenumerous references describing such methodology, e.g., U.S. Pat. No.4,671,958, to Rodwell et al.

In other illustrative embodiments, the conjugates include linking groupssuch as those disclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425235 B1, and Chari et al., Cancer Research 52: 127-131 (1992).Illustrative linking groups include, for example, disufide groups,thioether groups, acid labile groups, photolabile groups, peptidaselabile groups and esterase labile groups.

In still other illustrative embodiments, conjugates are made usingbifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCL), active esters (such as disuccinimidylsuberate), aldehydes (such as glutareldehyde), bis-azido compounds (suchas bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). Particular coupling agents includeN-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (Carlsson et al.,Biochem. J. 173:723-737 [1978]) andN-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for adisulfide linkage. The linker may be a “cleavable linker” facilitatingrelease of one or more cleavable components. For example, an acid-labilelinker may be used (Cancer Research 52: 127-131 (1992); U.S. Pat. No.5,208,020).

In other embodiments, non-proteinaceous polymers are used in a linkerfor coupling a p97 polypeptide sequence to an antibody, such as aHer2/neu antibody. These may include, for example, polyethylene glycol,polypropylene glycol, polyoxyalkylenes, or copolymers of polyethyleneglycol, polypropylene glycol, and the like.

Where one component of a conjugate may be more potent when free from theconjugate, it may be desirable to use a linker group which is cleavableduring or upon internalization into a cell. A number of differentcleavable linker groups have been described. The mechanisms for theintracellular release of an agent from these linker groups includecleavage by reduction of a disulfide bond (e.g., U.S. Pat. No.4,489,710, to Spitler), by irradiation of a photolabile bond (e.g., U.S.Pat. No. 4,625,014, to Senter et al.), by hydrolysis of derivatizedamino acid side chains (e.g., U.S. Pat. No. 4,638,045, to Kohn et al.),by serum complement-mediated hydrolysis (e.g., U.S. Pat. No. 4,671,958,to Rodwell et al.), and acid-catalyzed hydrolysis (e.g., U.S. Pat. No.4,569,789, to Blattler et al.).

Certain embodiments may employ one or more aldehyde tags to facilitateconjugation between a p97 polypeptide and an antibody or antigen-bindingfragment thereof (see U.S. Pat. Nos. 8,097,701 and 7,985,783,incorporated by reference). Here, enzymatic modification at a sulfatasemotif of the aldehyde tag through action of a formylglycine generatingenzyme (FGE) generates a formylglycine (FGly) residue. The aldehydemoiety of the FGly residue can then be exploited as a chemical handlefor site-specific attachment of a moiety of interest to the polypeptide.In some aspects, the moiety of interest is another polypeptide, such asan antibody.

Particular embodiments thus include a p97 polypeptide or antibody orantigen-binding fragment (e.g., anti-Her2/neu antibody) that comprises1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more heterologous sulfatase motifs,where the motif comprises the following structure:

(SEQ ID NO: 6) X₁Z₁X₂Z₂X₃

where Z₁ is cysteine or serine; Z₂ is a proline or alanine residue; X₁is present or absent and, when present, is any amino acid, where X₁ ispreferably present when the heterologous sulfatase motif is at anN-terminus of the aldehyde tagged polypeptide; and X₂ and X₃ are eachindependently any amino acid.

Polypeptides with the above-described motif can be modified by an FGEenzyme to generate a motif having a FGly residue, which, as noted above,can then be used for site-specific attachment of a second polypeptide,for instance, via a linker moiety. Such modifications can be performed,for example, by expressing the sulfatase motif-containing polypeptide(e.g., p97, antibody) in a mammalian, yeast, or bacterial cell thatexpresses an FGE enzyme or by in vitro modification of isolatedpolypeptide with an isolated FGE enzyme (see Wu et al., PNAS.106:3000-3005, 2009; Rush and Bertozzi, J. Am Chem Soc. 130:12240-1,2008; and Carlson et al., J Biol Chem. 283:20117-25, 2008).

Hence, some embodiments include a p97 polypeptide or antibody (orantigen-binding fragment) that comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or more heterologous sulfatase motifs having a formylglycine residue,where the motif comprises the following structure:

(SEQ ID NO: 5) X₁(FGly)X₂Z₂X₃

where FGly is a formylglycine residue; Z₂ is a proline or alanineresidue; X₁ is present or absent and, when present, is any amino acid,where X₁ is preferably present when the heterologous sulfatase motif isat an N-terminus of the aldehyde tagged polypeptide; and X₂ and X₃ areeach independently any amino acid.

In particular embodiments, X₁, X₂, and X₃ are each independently analiphatic amino acid, a sulfur-containing amino acid or a polar,uncharged amino acid. For instance, X₁ can be L, M, V, S or T; and X₂,and/or X₃ can be independently S, T, A, V, G or C.

In some embodiments, the heterologous sulfatase motif(s) can be (a) lessthan 16 amino acid residues in length, including about 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, or 15 residues in length, (b) positioned at theN-terminus of the polypeptide, (c) positioned at the C-terminus of thepolypeptide, (d) positioned at an internal site of an amino acidsequence native to the polypeptide, (e) positioned in a terminal loop ofthe polypeptide, (f) positioned at a site of post-translationalmodification of the polypeptide (e.g., glycosylation site), or anycombination thereof. In specific embodiments, the antibody thatcomprises one or more heterologous sulfatase motif(s) specifically bindsto human Her2/neu (e.g.,. Trastuzumab).

Some embodiments relate to conjugates of (i) a sulfatase motif (oraldehyde tag)-containing p97 polypeptide, and (ii) an antibody orantigen-binding fragment thereof that is functionalized with an aldehydereactive group, or vice versa, where (i) and (ii) are covalently linkedvia the FGly residue of the sulfatase motif and the aldehyde reactivegroup. Such conjugates can have one of the following general structures:

p97(FGly)-R₁-Ab or p97-R₁-(FGly)Ab

where R₁ is at least one aldehyde reactive linkage; and FGly is aformylglycine residue within a heterologous sulfatase.

The non-aldehyde tag-containing protein (e.g., antibody, p97polypeptide) can be functionalized with one or more aldehyde reactivegroups such as aminooxy, hydrazide, and thiosemicarbazide, and thencovalently linked to the aldehyde tag-containing polypeptide via the atleast one FGly residue, to form an aldehyde reactive linkage. Theattachment of an aminooxy functionalized protein creates an oximelinkage between the FGly residue and the functionalized protein;attachment of a hydrazide-functionalized protein creates a hydrazinelinkage between the FGly residue and the functionalized protein; andattachment of a thiosemicarbazide-functionalized protein creates ahydrazine carbothiamide linkage between the FGly residue and thefunctionalized protein.

Certain embodiments include conjugates of (i) a sulfatase motif (oraldehyde tag)-containing p97 polypeptide and (ii) a sulfatase motif (oraldehyde tag)-containing antibody, where (i) and (ii) are covalentlylinked via their respective FGly residues, optionally by abi-functionalized linker moiety. For instance, certain p97-antibodyconjugates may comprise the following structure:

p97(FGly)-R₁-L-R₂-(FGly)Ab

where R₁ and R₂ are the same or different aldehyde reactive linkage; Lis a linker moiety, p97(FGly) is a aldehyde-tag containing p97polypeptide, and (FGly)Ab is an aldehyde tag-containing antibody, suchas an antibody that specifically binds to human Her2/neu (e.g.,Trastuzumab). Merely by way of illustration, in some embodiments, the atleast one heterologous sulfatase motif can be at the C-terminus of thep97 polypeptide and the N-terminus of the antibody. In otherembodiments, the at least one heterologous sulfatase motif can be at theN-terminus of the p97 polypeptide and the C-terminus of the antibody. Instill other embodiments, the at least one heterologous sulfatase motifcan be at the N-terminus of the p97 polypeptide and the N-terminus ofthe antibody. In further embodiments, the at least one heterologoussulfatase motif can be at the C-terminus of the p97 polypeptide an theC-terminus of the antibody. As noted above, the at least oneheterologous motif can be at an internal position in the p97 polypeptideand/or the antibody. Persons skilled in the art will recognize thatother combinations are possible.

The aldehyde reactive linkages of R₁ and R₂ can be independently formedby any aldehyde reactive group that will form a covalent bond between(i) the formylglycine (FGly) residue of the aldehyde tag and (ii) alinker moiety that is functionalized with said aldehyde reactive group(e.g., a bi-functionalized linker with two aldehyde reactive groups,which can be the same or different). Examples of aldehyde reactivegroups include aminooxy, hydrazide, and thiosemicarbazide groups, whichwill form Schiff-base containing linkages with a FGly residue, includingoxime linkages, hydrazine linkages, and hydrazine carbothiamidelinkages, respectively. Hence, R₁ and R₂ can be independently a linkagethat comprises a Schiff base, such as an oxime linkage, a hydrazinelinkage, or a hydrazine carbothiamide linkage.

In some embodiments, the aldehyde tag-containing p97 polypeptide and thealdehyde tag-containing antibody are linked (e.g., covalently linked)via a multi-functionalized linker (e.g., bi-functionalized linker), thelatter being functionalized with the same or different aldehyde reactivegroup(s). In these and related embodiments, the aldehyde reactive groupsallow the linker to form a covalent bridge between the p97 polypeptideand the antibody via their respective FGly residues. Linker moietiesinclude any moiety or chemical that can be functionalized and preferablybi- or multi-functionalized with one or more aldehyde reactive groups.Particular examples include peptides, water-soluble polymers, detectablelabels, other therapeutic compounds (e.g., cytotoxic compounds),biotin/streptavidin moieties, and glycans (see Hudak et al., J Am ChemSoc. 133:16127-35, 2011). Specific examples of glycans (or glycosides)include aminooxy glycans, such as higher-order glycans composed ofglycosyl N-pentenoyl hydroxamates intermediates (supra).

Peptide linkers (or spacers) are described below. Peptide linkers can befunctionalized with aldehyde reactive groups according to routinetechniques in the art (see, e.g., Carrico et al., Nat Chem Biol.3:321-322, 2007).

A “water-soluble polymer” refers to a polymer that is soluble in waterand is usually substantially non-immunogenic, and usually has an atomicmolecular weight greater than about 1,000 Daltons. Attachment of twopolypeptides via a water-soluble polymer can be desirable as suchmodification(s) can increase the therapeutic index by increasing serumhalf-life, for instance, by increasing proteolytic stability and/ordecreasing renal clearance. Additionally, attachment via of one or morepolymers can reduce the immunogenicity of protein pharmaceuticals.

In some embodiments, the water-soluble polymer has an effectivehydrodynamic molecular weight of greater than about 10,000 Da, greaterthan about 20,000 to 500,000 Da, greater than about 40,000 Da to 300,000Da, greater than about 50,000 Da to 70,000 Da, usually greater thanabout 60,000 Da. The “effective hydrodynamic molecular weight” refers tothe effective water-solvated size of a polymer chain as determined byaqueous-based size exclusion chromatography (SEC). When thewater-soluble polymer contains polymer chains having polyalkylene oxiderepeat units, such as ethylene oxide repeat units, each chain can havean atomic molecular weight of between about 200 Da and about 80,000 Da,or between about 1,500 Da and about 42,000 Da, with 2,000 to about20,000 Da being of particular interest. Linear, branched, and terminallycharged water soluble polymers are also included.

Polymers useful as linkers between aldehyde tagged polypeptides can havea wide range of molecular weights, and polymer subunits. These subunitsmay include a biological polymer, a synthetic polymer, or a combinationthereof. Examples of such water-soluble polymers include: dextran anddextran derivatives, including dextran sulfate, P-amino cross linkeddextrin, and carboxymethyl dextrin, cellulose and cellulose derivatives,including methylcellulose and carboxymethyl cellulose, starch anddextrines, and derivatives and hydroylactes of starch, polyalklyeneglycol and derivatives thereof, including polyethylene glycol (PEG),methoxypolyethylene glycol, polyethylene glycol homopolymers,polypropylene glycol homopolymers, copolymers of ethylene glycol withpropylene glycol, wherein said homopolymers and copolymers areunsubstituted or substituted at one end with an alkyl group, heparin andfragments of heparin, polyvinyl alcohol and polyvinyl ethyl ethers,polyvinylpyrrolidone, aspartamide, and polyoxyethylated polyols, withthe dextran and dextran derivatives, dextrine and dextrine derivatives.It will be appreciated that various derivatives of the specificallydescribed water-soluble polymers are also included.

Water-soluble polymers are known in the art, particularly thepolyalkylene oxide-based polymers such as polyethylene glycol “PEG” (seePoly(ethylene glycol) Chemistry: Biotechnical and BiomedicalApplications, J. M. Harris, Ed., Plenum Press, New York, N.Y. (1992);and Poly(ethylene glycol) Chemistry and Biological Applications, J. M.Harris and S. Zalipsky, Eds., ACS (1997); and International PatentApplications: WO 90/13540, WO 92/00748, WO 92/16555, WO 94/04193, WO94/14758, WO 94/17039, WO 94/18247, WO 94/28937, WO 95/11924, WO96/00080, WO 96/23794, WO 98/07713, WO 98/41562, WO 98/48837, WO99/30727, WO 99/32134, WO 99/33483, WO 99/53951, WO 01/26692, WO95/13312, WO 96/21469, WO 97/03106, WO 99/45964, and U.S. Pat. Nos.4,179,337; 5,075,046; 5,089,261; 5,100,992; 5,134,192; 5,166,309;5,171,264; 5,213,891; 5,219,564; 5,275,838; 5,281,698; 5,298,643;5,312,808; 5,321,095; 5,324,844; 5,349,001; 5,352,756; 5,405,877;5,455,027; 5,446,090; 5,470,829; 5,478,805; 5,567,422; 5,605,976;5,612,460; 5,614,549; 5,618,528; 5,672,662; 5,637,749; 5,643,575;5,650,388; 5,681,567; 5,686,110; 5,730,990; 5,739,208; 5,756,593;5,808,096; 5,824,778; 5,824,784; 5,840,900; 5,874,500; 5,880,131;5,900,461; 5,902,588; 5,919,442; 5,919,455; 5,932,462; 5,965,119;5,965,566; 5,985,263; 5,990,237; 6,011,042; 6,013,283; 6,077,939;6,113,906; 6,127,355; 6,177,087; 6,180,095; 6,194,580; 6,214,966,incorporated by reference).

Exemplary polymers of interest include those containing a polyalkyleneoxide, polyamide alkylene oxide, or derivatives thereof, includingpolyalkylene oxide and polyamide alkylene oxide comprising an ethyleneoxide repeat unit of the formula —(CH₂—CH²—O)—. Further exemplarypolymers of interest include a polyamide having a molecular weightgreater than about 1,000 Daltons of the formula—[C(O)—X—C(O)—NH—Y—NH]_(n)— or —[NH—Y—NH—C(O)—X—C(O)]_(n)—, where X andY are divalent radicals that may be the same or different and may bebranched or linear, and n is a discrete integer from 2-100, usually from2 to 50, and where either or both of X and Y comprises a biocompatible,substantially non-antigenic water-soluble repeat unit that may be linearor branched.

Further exemplary water-soluble repeat units comprise an ethylene oxideof the formula —(CH₂—CH₂—O)— or —(CH₂—CH₂—O)—. The number of suchwater-soluble repeat units can vary significantly, with the usual numberof such units being from 2 to 500, 2 to 400, 2 to 300, 2 to 200, 2 to100, and most usually 2 to 50. An exemplary embodiment is one in whichone or both of X and Y is selected from:—((CH₂)_(n1)—(CH₂—CH₂—O)_(n2)—(CH₂)— or—((CH₂)_(n1)—(O—CH₂—CH₂)_(n2)—(CH₂)_(n1)—), where n1 is 1 to 6, 1 to 5,1 to 4 and most usually 1 to 3, and where n2 is 2 to 50, 2 to 25, 2 to15, 2 to 10, 2 to 8, and most usually 2 to 5. A further exemplaryembodiment is one in which X is —(CH₂—CH₂)—, and where Y is—(CH₂—(CH₂—CH₂—O)₃—CH₂—CH₂—CH₂)— or —(CH₂—CH₂—CH₂—(O—CH₂—CH₂)₃—CH₂)—,among other variations.

For biotin/streptavidin (or avidin) moieties, the aldehydetag(s)-containing p97 polypeptide can be covalently attached via a FGlyresidue to a biotin molecule that is functionalized with an aldehydereactive group, and the aldehyde tag(s)-containing antibody can becovalently attached via a FGly residue to a streptavidin molecule thatis functionalized with an aldehyde reactive group, or vice versa. Thep97-biotin (or streptavidin) can then be mixed with theantibody-streptavidin (or biotin) to form a p97-antibody conjugate viathe strong interaction between biotin and streptavidin.

p97-antibody conjugates can also be prepared by a various “clickchemistry” techniques, including reactions that are modular, wide inscope, give very high yields, generate mainly inoffensive byproductsthat can be removed by non-chromatographic methods, and can bestereospecific but not necessarily enantioselective (see Kolb et al.,Angew Chem Int Ed Engl. 40:2004-2021, 2001). Particular examples includeconjugation techniques that employ the Huisgen 1,3-dipolar cycloadditionof azides and alkynes, also referred to as “azide-alkyne cycloaddition”reactions (see Hein et al., Pharm Res. 25:2216-2230, 2008). Non-limitingexamples of azide-alkyne cycloaddition reactions includecopper-catalyzed azide-alkyne cycloaddition (CuAAC) reactions andruthenium-catalyzed azide-alkyne cycloaddition (RuAAC) reactions.

CuAAC works over a broad temperature range, is insensitive to aqueousconditions and a pH range over 4 to 12, and tolerates a broad range offunctional groups (see Himo et al, J Am Chem Soc. 127:210-216, 2005).The active Cu(I) catalyst can be generated, for example, from Cu(I)salts or Cu(II) salts using sodium ascorbate as the reducing agent. Thisreaction forms 1,4-substituted products, making it region-specific (seeHein et al., supra).

RuAAC utilizes pentamethylcyclopentadienyl ruthenium chloride [Cp*RuCl]complexes that are able to catalyze the cycloaddition of azides toterminal alkynes, regioselectively leading to 1,5-disubstituted1,2,3-triazoles (see Rasmussen et al., Org. Lett. 9:5337-5339, 2007).Further, and in contrast to CuAAC, RuAAC can also be used with internalalkynes to provide fully substituted 1,2,3-triazoles.

Certain embodiments thus include p97 polypeptides that comprise at leastone unnatural amino acid with an azide side-chain or an alkyneside-chain, including internal and terminal unnatural amino acids (e.g.,N-terminal, C-terminal). Certain of these p97 polypeptides can be formedby in vivo or in vitro (e.g., cell-free systems) incorporation ofunnatural amino acids that contain azide side-chains or alkyneside-chains. Exemplary in vivo techniques include cell culturetechniques, for instance, using modified E. coli (see Travis andSchultz, The Journal of Biological Chemistry. 285:11039-44, 2010; andDeiters and Schultz, Bioorganic & Medicinal Chemistry Letters.15:1521-1524, 2005), and exemplary in vitro techniques include cell-freesystems (see Bundy, Bioconjug Chem. 21:255-63, 2010).

In some embodiments, a p97 polypeptide that comprises at least oneunnatural amino acid with an azide side-chain is conjugated byazide-alkyne cycloaddition to an antibody that comprises at least oneunnatural amino acid with an alkyne side-chain. In other embodiments, ap97 polypeptide that comprises at least one unnatural amino acid with analkyne side-chain is conjugated by azide-alkyne cycloaddition to anantibody that comprises at least one unnatural amino acid with an azideside-chain. Hence, certain embodiments include conjugates that comprisea p97 polypeptide covalently linked to an antibody via a 1,2,3-triazolelinkage. In specific embodiments, the antibody is an anti-Her2/neuantibody such as Trastuzumab.

Specific p97-antibody conjugates can be formed by the followingCuAAC-based or RuAAC-based reactions, to comprise the followingrespective structures (I) or (II):

where R is a p97 polypeptide and R¹ is an antibody or antigen-bindingfragment thereof; or where R is an antibody or antigen-binding fragmentthereof and R¹ is a p97 polypeptide.

As noted above, in some embodiments the unnatural amino acid with theazide side-chain and/or the unnatural amino acid with alkyne side-chainare terminal amino acids (N-terminal, C-terminal). In certainembodiments, one or more of the unnatural amino acids are internal.Specific embodiments include a p97 polypeptide that comprises anN-terminal unnatural amino acid with an azide side-chain conjugated toan antibody that comprises an N-terminal unnatural amino acid with analkyne side-chain. Other embodiments include a p97 polypeptide thatcomprises a C-terminal unnatural amino acid with an azide side-chainconjugated to an antibody that comprises a C-terminal unnatural aminoacid with an alkyne side-chain. Still other embodiments include a p97polypeptide that comprises an N-terminal unnatural amino acid with anazide side-chain conjugated to an antibody that comprises a C-terminalunnatural amino acid with an alkyne side-chain. Further embodimentsinclude a p97 polypeptide that comprises a C-terminal unnatural aminoacid with an azide side-chain conjugated to an antibody that comprisesan N-terminal unnatural amino acid with an alkyne side-chain.

Other embodiments include a p97 polypeptide that comprises an N-terminalunnatural amino acid with an alkyne side-chain conjugated to an antibodythat comprises an N-terminal unnatural amino acid with an azideside-chain. Still further embodiments include a p97 polypeptide thatcomprises a C-terminal unnatural amino acid with an alkyne side-chainconjugated to an antibody that comprises a C-terminal unnatural aminoacid with an azide side-chain. Additional embodiments include a p97polypeptide that comprises an N-terminal unnatural amino acid with analkyne side-chain conjugated to an antibody that comprises a C-terminalunnatural amino acid with an azide side-chain. Still further embodimentsinclude a p97 polypeptide that comprises a C-terminal unnatural aminoacid with an alkyne side-chain conjugated to an antibody that comprisesan N-terminal unnatural amino acid with an azide side-chain.

Also included are methods of producing a p97-antibody conjugate,comprising: (a) performing an azide-alkyne cycloaddition reactionbetween (i) a p97 polypeptide that comprises at least one unnaturalamino acid with an azide side-chain and an antibody or antigen-bindingfragment thereof that comprises at least one unnatural amino acid withan alkyne side-chain; or (ii) a p97 polypeptide that comprises at leastone unnatural amino acid with an alkyne side-chain and an antibody orantigen-binding fragment thereof that comprises at least one unnaturalamino acid with an azide side-chain; and (b) isolating a p97-antibodyconjugate from the reaction, thereby producing a p97-antibody conjugate.

In the case where the p97-antibody conjugate is a fusion polypeptide,the fusion polypeptide may generally be prepared using standardtechniques, including chemical conjugation. Preferably, however, afusion polypeptide is expressed as a recombinant polypeptide in anexpression system, as described below. Fusion polypeptides of theinvention can contain one or multiple copies of a p97 polypeptidesequence and may contain one or multiple copies of an antibody orantigen-binding fragment thereof (e.g., anti-Her2/neu antibody orantigen-binding fragment thereof), present in any desired arrangement.

A peptide linker sequence may be employed to separate first and secondpolypeptide components by a distance sufficient to ensure that eachpolypeptide folds into its secondary and tertiary structures. Such apeptide linker sequence may be incorporated into the fusion polypeptideusing standard techniques well known in the art. Suitable peptide linkersequences may be chosen based on the following factors: (1) theirability to adopt a flexible extended conformation; (2) their inabilityto adopt a secondary structure that could interact with functionalepitopes on the first and second polypeptides; and (3) the lack ofhydrophobic or charged residues that might react with the polypeptidefunctional epitopes. Amino acid sequences which may be usefully employedas linkers include those disclosed in Maratea et al., Gene 40:39-46,1985; Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258-8262, 1986; U.S.Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180. The linker sequence maygenerally be from 1 to about 50 amino acids in length. Linker sequencesare not required when the first and second polypeptides havenon-essential N-terminal amino acid regions that can be used to separatethe functional domains and prevent steric interference.

In certain illustrative embodiments, a peptide spacer is between 1 to 5amino acids, between 5 to 10 amino acids, between 5 to 25 amino acids,between 5 to 50 amino acids, between 10 to 25 amino acids, between 10 to50 amino acids, between 10 to 100 amino acids, or any intervening rangeof amino acids. In other illustrative embodiments, a peptide spacercomprises about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more aminoacids in length. Particular linkers can have an overall amino acidlength of about 1-200 amino acids, 1-150 amino acids, 1-100 amino acids,1-90 amino acids, 1-80 amino acids, 1-70 amino acids, 1-60 amino acids,1-50 amino acids, 1-40 amino acids, 1-30 amino acids, 1-20 amino acids,1-10 amino acids, 1-5 amino acids, 1-4 amino acids, 1-3 amino acids, orabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100or more amino acids.

A peptide linker may employ any one or more naturally-occurring aminoacids, non-naturally occurring amino acid(s), amino acid analogs, and/oramino acid mimetics as described elsewhere herein and known in the art.Certain amino acid sequences which may be usefully employed as linkersinclude those disclosed in Maratea et al., Gene 40:39-46, 1985; Murphyet al., PNAS USA. 83:8258-8262, 1986; U.S. Pat. No. 4,935,233 and U.S.Pat. No. 4,751,180. Particular peptide linker sequences contain Gly,Ser, and/or Asn residues. Other near neutral amino acids, such as Thrand Ala may also be employed in the peptide linker sequence, if desired.

Certain exemplary linkers include Gly, Ser and/or Asn-containinglinkers, as follows: [G]_(x), [S]_(x), [N]_(x), [GS]_(x), [GGS]_(x),[GSS]_(x), [GSGS]_(x)(SEQ ID NO:7), [GGSG]_(x)(SEQ ID NO:8),[GGGS]_(x)(SEQ ID NO:9), [GGGGS]_(x) (SEQ ID NO:10), [GN]_(X),[GGN]_(x), [GNN]_(x), [GNGN]_(x) (SEQ ID NO:11), [GGNG]_(x) (SEQ IDNO:12), [GGGN]_(x)(SEQ ID NO:13), [GGGGN]_(x) (SEQ ID NO:14) linkers,where x is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, or 20 or more. Other combinations of these and related aminoacids will be apparent to persons skilled in the art.

In specific embodiments, the linker sequence comprises a Gly3 linkersequence, which includes three glycine residues. In particularembodiments, flexible linkers can be rationally designed using acomputer program capable of modeling both DNA-binding sites and thepeptides themselves (Desjarlais & Berg, PNAS. 90:2256-2260, 1993; andPNAS. 91:11099-11103, 1994) or by phage display methods.

The peptide linkers may be physiologically stable or may include areleasable linker such as a physiologically degradable or enzymaticallycleavable linker (e.g., protealytically cleavable linker). In certainembodiments, one or more releasable linkers can result in a shorterhalf-life and more rapid clearance of the conjugate. These and relatedembodiments can be used, for example, to enhance the solubility andblood circulation lifetime of p97-antibody conjugates in thebloodstream, while also delivering an antibody into the bloodstream (oracross the BBB) that, subsequent to linker degradation, is substantiallyfree of the p97 sequence. These aspects are especially useful in thosecases where antibodies, when permanently conjugated to a p97 sequence,demonstrate reduced activity. By using the linkers as provided herein,such antibodies can maintain their therapeutic activity when inconjugated form. In these and other ways, the properties of thep97-antibody conjugates can be more effectively tailored to balance thebioactivity and circulating half-life of the antibodies over time.

In particular embodiments, a releasable linker has a half life at pH7.4, 25° C., e.g., a physiological pH, human body temperature (e.g., invivo, in serum, in a given tissue), of about 30 minutes, about 1 hour,about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours,about 48 hours, about 72 hours, or about 96 hours or more or anyintervening half-life. One having skill in the art would appreciate thatthe half life of a p97-antibody conjugate can be finely tailored byusing a particular releasable linker.

In certain embodiments, however, any one or more of the peptide linkersare optional. For instance, linker sequences may not required when thefirst and second polypeptides have non-essential N-terminal and/orC-terminal amino acid regions that can be used to separate thefunctional domains and prevent steric interference.

In other embodiments, a p97 antibody conjugate as described herein maybe further conjugated or operably linked to another therapeuticcompound. The conjugate may include, for example, a cytotoxic agent, achemotherapeutic agent, a cytokine, an anti-angiogenic agent, a tyrosinekinase inhibitor, a toxin, a radioisotope, or other therapeuticallyactive agent.

Fusion Polynucleotides, Host Cells and Recombinant Production

The present invention further provides in certain embodiments isolatedpolynucleotides encoding p97 polypeptides and 97-antibody conjugates ofthe invention, such as a fusion polypeptide comprising an anti-Her2/neuantibody or antigen binding fragment and a p97 polypeptide sequence orfragment or derivative thereof. Also included are isolated orrecombinant polynucleotides that encode aldehyde-tag containing p97polypeptides and antibodies, and p97 polypeptides and antibodies thatcomprise at least one unnatural amino acid, for instance, unnaturalamino acids with an azide side-chain or alkyne side-chain, and relatedhost cells. These and related embodiments can be used for therecombinant production of the p97-antibody fusion proteins andnon-fusion conjugates described herein.

For fusion proteins, DNA sequences encoding the p97 and antibody (e.g.,anti-Her2/neu antibody) components may be assembled separately, and thenligated into an appropriate expression vector. The 3′ end of the DNAsequence encoding one polypeptide component is ligated, with or withouta peptide linker, to the 5′ end of a DNA sequence encoding the secondpolypeptide component so that the reading frames of the sequences are inphase. The ligated DNA sequences are operably linked to suitabletranscriptional or translational regulatory elements. The regulatoryelements responsible for expression of DNA are located only 5′ to theDNA sequence encoding the first polypeptides. Similarly, stop codonsrequired to end translation and transcription termination signals areonly present 3′ to the DNA sequence encoding the second polypeptide.This permits translation into a single fusion polypeptide that retainsthe biological activity of both component polypeptides.

Similar techniques, mainly the arrangement of regulatory elements suchas promoters, stop codons, and transcription termination signals, can beapplied to the recombinant production of non-fusion proteins, forinstance, p97 polypeptides and antibodies for the production ofnon-fusion conjugates.

Polynucleotides and fusion polynucleotides of the invention can containone or multiple copies of a nucleic acid encoding a p97 polypeptidesequence, and/or may contain one or multiple copies of a nucleic acidencoding an antibody or antigen-binding fragment thereof.

In some embodiments, a nucleic acids encoding a subject p97 polypeptide,antibody, and/or p97-antibody fusion are introduced directly into a hostcell, and the cell incubated under conditions sufficient to induceexpression of the encoded polypeptide(s). The polypeptide sequences ofthis disclosure may be prepared using standard techniques well known tothose of skill in the art in combination with the polypeptide andnucleic acid sequences provided herein.

Therefore, according to certain related embodiments, there is provided arecombinant host cell which comprises a polynucleotide or a fusionpolynucleotide as described herein. Expression of a p97 polypeptide,antibody, or p97-antibody fusion in the host cell may conveniently beachieved by culturing under appropriate conditions recombinant hostcells containing the polynucleotide. Following production by expression,the polypeptide(s) may be isolated and/or purified using any suitabletechnique, and then used as desired.

Systems for cloning and expression of a polypeptide in a variety ofdifferent host cells are well known. Suitable host cells includebacteria, mammalian cells, yeast and baculovirus systems. Mammalian celllines available in the art for expression of a heterologous polypeptideinclude Chinese hamster ovary cells, HeLa cells, baby hamster kidneycells, HEK-293 cells, NSO mouse melanoma cells and many others. Acommon, preferred bacterial host is E. coli.

The expression of antibodies and antigen-binding fragments inprokaryotic cells such as E. coli is well established in the art. For areview, see for example Pluckthun, A. Bio/Technology. 9:545-551 (1991).Expression in eukaryotic cells in culture is also available to thoseskilled in the art as an option for production of antibodies orantigen-binding fragments thereof, see recent reviews, for example Ref,M. E. (1993) Curr. Opinion Biotech. 4: 573-576; Trill J. J. et al.(1995) Curr. Opinion Biotech 6: 553-560.

Suitable vectors can be chosen or constructed, containing appropriateregulatory sequences, including promoter sequences, terminatorsequences, polyadenylation sequences, enhancer sequences, marker genesand other sequences as appropriate. Vectors may be plasmids, viral e.g.phage, or phagemid, as appropriate. For further details see, forexample, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrooket al., 1989, Cold Spring Harbor Laboratory Press. Many known techniquesand protocols for manipulation of nucleic acid, for example inpreparation of nucleic acid constructs, mutagenesis, sequencing,introduction of DNA into cells and gene expression, and analysis ofproteins, are described in detail in Current Protocols in MolecularBiology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992,or subsequent updates thereto.

The term “host cell” is used to refer to a cell into which has beenintroduced, or which is capable of having introduced into it, a nucleicacid sequence encoding one or more of the herein described polypeptides,and which further expresses or is capable of expressing a selected geneof interest, such as a gene encoding any herein described polypeptide.The term includes the progeny of the parent cell, whether or not theprogeny are identical in morphology or in genetic make-up to theoriginal parent, so long as the selected gene is present. Host cells maybe chosen for certain characteristics, for instance, the expression of aformylglycine generating enzyme (FGE) to convert a cysteine or serineresidue within a sulfatase motif into a formylglycine (FGly) residue, orthe expression of aminoacyl tRNA synthetase(s) that can incorporateunnatural amino acids into the polypeptide, including unnatural aminoacids with an azide side-chain, alkyne side-chain, or other desiredside-chain, to facilitate conjugation.

Accordingly there is also contemplated a method comprising introducingsuch nucleic acid(s) into a host cell. The introduction may employ anyavailable technique. For eukaryotic cells, suitable techniques mayinclude calcium phosphate transfection, DEAE-Dextran, electroporation,liposome-mediated transfection and transduction using retrovirus orother virus, e.g. vaccinia or, for insect cells, baculovirus. Forbacterial cells, suitable techniques may include calcium chloridetransformation, electroporation and transfection using bacteriophage.The introduction may be followed by causing or allowing expression fromthe nucleic acid, e.g. by culturing host cells under conditions forexpression of the gene. In one embodiment, the nucleic acid isintegrated into the genome (e.g. chromosome) of the host cell.Integration may be promoted by inclusion of sequences which promoterecombination with the genome, in accordance-with standard techniques.

The present invention also provides, in certain embodiments, a methodwhich comprises using a construct as stated above in an expressionsystem in order to express a particular polypeptide, such as a p97polypeptide, antibody, or p97-antibody fusion protein as describedherein.

Illustratively, a peptide linker/spacer sequence may be employed toseparate the components of a p97-antibody fusion protein by a distancesufficient to ensure that each polypeptide folds into its secondaryand/or tertiary structures, if desired. Such a peptide linker sequencecan be incorporated into a fusion polypeptide using standard techniqueswell known in the art.

Compositions and Methods of Use

The present disclosure also provides compositions comprising thep97-antibody conjugates and compositions of the invention andadministration of such compositions for therapeutic purposes.

Administration of the conjugates described herein, in pure form or in anappropriate pharmaceutical composition, can be carried out via any ofthe accepted modes of administration of agents for serving similarutilities. The pharmaceutical compositions can be prepared by combininga conjugate or conjugate-containing composition with an appropriatephysiologically acceptable carrier, diluent or excipient, and may beformulated into preparations in solid, semi-solid, liquid or gaseousforms, such as tablets, capsules, powders, granules, ointments,solutions, suppositories, injections, inhalants, gels, microspheres, andaerosols. In addition, other pharmaceutically active ingredients(including other anti-cancer agents as described elsewhere herein)and/or suitable excipients such as salts, buffers and stabilizers may,but need not, be present within the composition. Administration may beachieved by a variety of different routes, including oral, parenteral,nasal, intravenous, intradermal, subcutaneous or topical. Preferredmodes of administration depend upon the nature of the condition to betreated or prevented. An amount that, following administration, reduces,inhibits, prevents or delays the progression and/or metastasis of acancer is considered effective.

Carriers can include, for example, pharmaceutically acceptable carriers,excipients, or stabilizers that are nontoxic to the cell or mammal beingexposed thereto at the dosages and concentrations employed. Often thephysiologically acceptable carrier is an aqueous pH buffered solution.Examples of physiologically acceptable carriers include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid; low molecular weight (less than about 10 residues)polypeptide; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, arginine or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; salt-forming counterions such assodium; and/or nonionic surfactants such as polysorbate 20 (TWEEN™)polyethylene glycol (PEG), and poloxamers (PLURONICS™) and the like.

The present invention provides therapeutic compositions comprising a p97polypeptide sequence and any therapeutic and/or diagnostic antibody orantigen-binding fragment thereof (e.g., an antibody or fragment thatspecifically binds the human Her2/neu protein or other antibodydescribed herein).

In certain aspects, the p97 polypeptide sequence and the antibody orfragment thereof are each, individually or as a pre-existing conjugate,bound to or encapsulated within a particle, e.g., a nanoparticle, bead,lipid formulation, lipid particle, or liposome, e.g., immunoliposome.For instance, in particular embodiments, the p97 polypeptide sequence isbound to the surface of a particle, and the antibody or fragment thereofis bound to the surface of the particle and/or encapsulated within theparticle. In certain of these and related embodiments, the p97polypeptide and the antibody are covalently or operatively linked toeach other only via the particle itself (e.g., nanoparticle, liposome),and are not covalently linked to each other in any other way; that is,they are bound individually to the same particle. In other embodiments,the p97 polypeptide and the antibody are first covalently conjugated toeach other, as described herein (e.g., via a linker molecule), and arethen bound to or encapsulated within a particle (e.g., immunoliposome,nanoparticle). In specific embodiments, the particle is a liposome, andthe composition comprises one or more p97 polypeptides, one or moreantibodies or antigen-binding fragments thereof, and a mixture of lipidsto form a liposome (e.g., phospholipids, mixed lipid chains withsurfactant properties). In some aspects, the p97 polypeptide and theantibody (or antigen-binding fragment) are individually mixed with thelipid/liposome mixture, such that the formation of liposome structuresoperatively links the p97 polypeptide and antibody without the need forcovalent conjugation. In other aspects, the p97 polypeptide and theantibody (or antigen-binding fragment) are first covalently conjugatedto each other, as described herein, and then mixed with lipids to form aliposome. The p97 polypeptide, the antibody, or the p97antibody-conjugate may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization(for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate)microcapsules, respectively), in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules), or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,16th edition, Oslo, A., Ed., (1980). The particle(s) or liposomes mayfurther comprise other cytotoxic agents.

The precise dosage and duration of treatment is a function of thedisease being treated and may be determined empirically using knowntesting protocols or by testing the compositions in model systems knownin the art and extrapolating therefrom. Controlled clinical trials mayalso be performed. Dosages may also vary with the severity of thecondition to be alleviated. A pharmaceutical composition is generallyformulated and administered to exert a therapeutically useful effectwhile minimizing undesirable side effects. The composition may beadministered one time, or may be divided into a number of smaller dosesto be administered at intervals of time. For any particular subject,specific dosage regimens may be adjusted over time according to theindividual need.

The conjugate-containing compositions may be administered alone or incombination with other known cancer treatments, such as radiationtherapy, chemotherapy, transplantation, immunotherapy, hormone therapy,photodynamic therapy, etc. The compositions may also be administered incombination with antibiotics.

Typical routes of administering these and related pharmaceuticalcompositions thus include, without limitation, oral, topical,transdermal, inhalation, parenteral, sublingual, buccal, rectal,vaginal, and intranasal. The term parenteral as used herein includessubcutaneous injections, intravenous, intramuscular, intrasternalinjection or infusion techniques. Pharmaceutical compositions accordingto certain embodiments of the present invention are formulated so as toallow the active ingredients contained therein to be bioavailable uponadministration of the composition to a patient. Compositions that willbe administered to a subject or patient may take the form of one or moredosage units, where for example, a tablet may be a single dosage unit,and a container of a herein described conjugate in aerosol form may holda plurality of dosage units. Actual methods of preparing such dosageforms are known, or will be apparent, to those skilled in this art; forexample, see Remington: The Science and Practice of Pharmacy, 20thEdition (Philadelphia College of Pharmacy and Science, 2000). Thecomposition to be administered will, in any event, contain atherapeutically effective amount of a conjugate of the presentdisclosure, for treatment of a disease or condition of interest inaccordance with teachings herein.

A pharmaceutical composition may be in the form of a solid or liquid. Inone embodiment, the carrier(s) are particulate, so that the compositionsare, for example, in tablet or powder form. The carrier(s) may beliquid, with the compositions being, for example, an oral oil,injectable liquid or an aerosol, which is useful in, for example,inhalatory administration. When intended for oral administration, thepharmaceutical composition is preferably in either solid or liquid form,where semi-solid, semi-liquid, suspension and gel forms are includedwithin the forms considered herein as either solid or liquid.

As a solid composition for oral administration, the pharmaceuticalcomposition may be formulated into a powder, granule, compressed tablet,pill, capsule, chewing gum, wafer or the like. Such a solid compositionwill typically contain one or more inert diluents or edible carriers. Inaddition, one or more of the following may be present: binders such ascarboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, gumtragacanth or gelatin; excipients such as starch, lactose or dextrins,disintegrating agents such as alginic acid, sodium alginate, Primogel,corn starch and the like; lubricants such as magnesium stearate orSterotex; glidants such as colloidal silicon dioxide; sweetening agentssuch as sucrose or saccharin; a flavoring agent such as peppermint,methyl salicylate or orange flavoring; and a coloring agent. When thepharmaceutical composition is in the form of a capsule, for example, agelatin capsule, it may contain, in addition to materials of the abovetype, a liquid carrier such as polyethylene glycol or oil.

The pharmaceutical composition may be in the form of a liquid, forexample, an elixir, syrup, solution, emulsion or suspension. The liquidmay be for oral administration or for delivery by injection, as twoexamples. When intended for oral administration, preferred compositioncontain, in addition to the present compounds, one or more of asweetening agent, preservatives, dye/colorant and flavor enhancer. In acomposition intended to be administered by injection, one or more of asurfactant, preservative, wetting agent, dispersing agent, suspendingagent, buffer, stabilizer and isotonic agent may be included.

The liquid pharmaceutical compositions, whether they be solutions,suspensions or other like form, may include one or more of the followingadjuvants: sterile diluents such as water for injection, salinesolution, preferably physiological saline, Ringer's solution, isotonicsodium chloride, fixed oils such as synthetic mono or diglycerides whichmay serve as the solvent or suspending medium, polyethylene glycols,glycerin, propylene glycol or other solvents; antibacterial agents suchas benzyl alcohol or methyl paraben; antioxidants such as ascorbic acidor sodium bisulfite; chelating agents such as ethylenediaminetetraaceticacid; buffers such as acetates, citrates or phosphates and agents forthe adjustment of tonicity such as sodium chloride or dextrose. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic. Physiological saline isa preferred adjuvant. An injectable pharmaceutical composition ispreferably sterile.

A liquid pharmaceutical composition intended for either parenteral ororal administration should contain an amount of a conjugate as hereindisclosed such that a suitable dosage will be obtained. Typically, thisamount is at least 0.01% of the antibody in the composition. Whenintended for oral administration, this amount may be varied to bebetween 0.1 and about 70% of the weight of the composition. Certain oralpharmaceutical compositions contain between about 4% and about 75% ofthe antibody. In certain embodiments, pharmaceutical compositions andpreparations according to the present invention are prepared so that aparenteral dosage unit contains between 0.01 to 10% by weight of theantibody prior to dilution.

The pharmaceutical composition may be intended for topicaladministration, in which case the carrier may suitably comprise asolution, emulsion, ointment or gel base. The base, for example, maycomprise one or more of the following: petrolatum, lanolin, polyethyleneglycols, bee wax, mineral oil, diluents such as water and alcohol, andemulsifiers and stabilizers. Thickening agents may be present in apharmaceutical composition for topical administration. If intended fortransdermal administration, the composition may include a transdermalpatch or iontophoresis device. The pharmaceutical composition may beintended for rectal administration, in the form, for example, of asuppository, which will melt in the rectum and release the drug. Thecomposition for rectal administration may contain an oleaginous base asa suitable nonirritating excipient. Such bases include, withoutlimitation, lanolin, cocoa butter and polyethylene glycol.

The pharmaceutical composition may include various materials, whichmodify the physical form of a solid or liquid dosage unit. For example,the composition may include materials that form a coating shell aroundthe active ingredients. The materials that form the coating shell aretypically inert, and may be selected from, for example, sugar, shellac,and other enteric coating agents. Alternatively, the active ingredientsmay be encased in a gelatin capsule. The pharmaceutical composition insolid or liquid form may include an agent that binds to the antibody ofthe invention and thereby assists in the delivery of the compound.Suitable agents that may act in this capacity include other monoclonalor polyclonal antibodies, one or more proteins or a liposome. Thepharmaceutical composition may consist essentially of dosage units thatcan be administered as an aerosol. The term aerosol is used to denote avariety of systems ranging from those of colloidal nature to systemsconsisting of pressurized packages. Delivery may be by a liquefied orcompressed gas or by a suitable pump system that dispenses the activeingredients. Aerosols may be delivered in single phase, bi-phasic, ortri-phasic systems in order to deliver the active ingredient(s).Delivery of the aerosol includes the necessary container, activators,valves, subcontainers, and the like, which together may form a kit. Oneof ordinary skill in the art, without undue experimentation maydetermine preferred aerosols.

The pharmaceutical compositions may be prepared by methodology wellknown in the pharmaceutical art. For example, a pharmaceuticalcomposition intended to be administered by injection can be prepared bycombining a composition that comprises a conjugate as described hereinand optionally, one or more of salts, buffers and/or stabilizers, withsterile, distilled water so as to form a solution. A surfactant may beadded to facilitate the formation of a homogeneous solution orsuspension. Surfactants are compounds that non-covalently interact withthe antibody composition so as to facilitate dissolution or homogeneoussuspension of the antibody in the aqueous delivery system.

The compositions may be administered in a therapeutically effectiveamount, which will vary depending upon a variety of factors includingthe activity of the specific compound (e.g., conjugate) employed; themetabolic stability and length of action of the compound; the age, bodyweight, general health, sex, and diet of the patient; the mode and timeof administration; the rate of excretion; the drug combination; theseverity of the particular disorder or condition; and the subjectundergoing therapy. Generally, a therapeutically effective daily dose is(for a 70 kg mammal) from about 0.001 mg/kg (i.e., 0.07 mg) to about 100mg/kg (i.e., 7.0 g); preferably a therapeutically effective dose is (fora 70 kg mammal) from about 0.01 mg/kg (i.e., 0.7 mg) to about 50 mg/kg(i.e., 3.5 g); more preferably a therapeutically effective dose is (fora 70 kg mammal) from about 1 mg/kg (i.e., 70 mg) to about 25 mg/kg(i.e., 1.75 g).

Compositions comprising the conjugates of the present disclosure mayalso be administered simultaneously with, prior to, or afteradministration of one or more other therapeutic agents. Such combinationtherapy may include administration of a single pharmaceutical dosageformulation which contains a compound of the invention and one or moreadditional active agents, as well as administration of compositionscomprising conjugates of the invention and each active agent in its ownseparate pharmaceutical dosage formulation. For example, a conjugate asdescribed herein and the other active agent can be administered to thepatient together in a single oral dosage composition such as a tablet orcapsule, or each agent administered in separate oral dosageformulations. Similarly, a conjugate as described herein and the otheractive agent can be administered to the patient together in a singleparenteral dosage composition such as in a saline solution or otherphysiologically acceptable solution, or each agent administered inseparate parenteral dosage formulations. Where separate dosageformulations are used, the compositions comprising conjugates and one ormore additional active agents can be administered at essentially thesame time, i.e., concurrently, or at separately staggered times, i.e.,sequentially and in any order; combination therapy is understood toinclude all these regimens.

Thus, in certain embodiments, also contemplated is the administration ofconjugate compositions of this disclosure in combination with one ormore other therapeutic agents. Such therapeutic agents may be acceptedin the art as a standard treatment for a particular disease state asdescribed herein, such as rheumatoid arthritis, inflammation or cancer.Exemplary therapeutic agents contemplated include cytokines, growthfactors, steroids, NSAIDs, DMARDs, anti-inflammatories,chemotherapeutics, radiotherapeutics, or other active and ancillaryagents.

In certain embodiments, the conjugates disclosed herein may beadministered in conjunction with any number of chemotherapeutic agents.Examples of chemotherapeutic agents include alkylating agents such asthiotepa and cyclophosphamide (CYTOXAN™); alkyl sulfonates such asbusulfan, improsulfan and piposulfan; aziridines such as benzodopa,carboquone, meturedopa, and uredopa; ethylenimines and methylamelaminesincluding altretamine, triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin,carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine,5-FU; androgens such as calusterone, dromostanolone propionate,epitiostanol, mepitiostane, testolactone; anti-adrenals such asaminoglutethimide, mitotane, trilostane; folic acid replenisher such asfrolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane;sizofiran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g.paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) anddoxetaxel (TAXOTERE®, Rhne-Poulenc Rorer, Antony, France); chlorambucil;gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinumanalogs such as cisplatin and carboplatin; vinblastine; platinum;etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine;vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin;xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000;difluoromethylomithine (DMFO); retinoic acid derivatives such asTargretin™ (bexarotene), Panretin™ (alitretinoin); ONTAK™ (denileukindiftitox); esperamicins; capecitabine; and pharmaceutically acceptablesalts, acids or derivatives of any of the above. Also included in thisdefinition are anti-hormonal agents that act to regulate or inhibithormone action on tumors such as anti-estrogens including for exampletamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles,4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, andtoremifene (Fareston); and anti-androgens such as flutamide, nilutamide,bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptablesalts, acids or derivatives of any of the above.

A variety of other therapeutic agents may be used in conjunction withthe conjugates described herein. In one embodiment, the conjugate isadministered with an anti-inflammatory agent. Anti-inflammatory agentsor drugs include, but are not limited to, steroids and glucocorticoids(including betamethasone, budesonide, dexamethasone, hydrocortisoneacetate, hydrocortisone, hydrocortisone, methylprednisolone,prednisolone, prednisone, triamcinolone), nonsteroidal anti-inflammatorydrugs (NSAIDS) including aspirin, ibuprofen, naproxen, methotrexate,sulfasalazine, leflunomide, anti-TNF medications, cyclophosphamide andmycophenolate.

The compositions may be administered to an individual afflicted with adisease as described herein, including, but not limited to neoplasticdiseases, metabolic diseases, neurological diseases, infections,cardiovascular diseases, inflammatory diseases, autoimmune diseases, anddiseases associated with abnormal angiogenesis. Particular diseasesinclude Her2/neu-expressing disorders, such as Her2/neu-expressingcancers.

Certain embodiments include methods of treating cancer in a subject,comprising administering to the subject a p97-antibody conjugatedescribed herein, or a composition comprising a p97-antibody conjugateand a pharmaceutically acceptable carrier or excipient. “Cancer” relatesgenerally to a class of diseases or conditions in which a group of cellsdisplay one or more of uncontrolled growth (i.e., division beyond normallimits), invasion (i.e., intrusion on and destruction of adjacenttissues), and/or metastasis (i.e., spread to other locations in the bodyvia lymph or blood). These malignant properties of cancers differentiatethem from benign cancers, which are self-limited, and typically do notinvade or metastasize.

A “cancer cell” or “tumor cell” refers to an individual cell of acancerous growth or tissue. A tumor refers generally to a swelling orlesion formed by an abnormal growth of cells, which may be benign,pre-malignant, or malignant. Most cancers form solid tumors, but some,e.g., leukemia, do not necessarily form tumors. For those cancers thatform tumors, the terms cancer (cell) and tumor (cell) are usedinterchangeably. General examples include primary and metastaticcancers. Particular examples of primary or metastatic cancers include,without limitation, prostate cancers, breast cancers, gastrointestinalcancers (e.g., colon cancers, colorectal carcinoma, rectal cancers),lung cancers (e.g., small lung cell cancers, non-small lung cellcarcinomas), ovarian cancers, testicular cancers, head and neck cancers,stomach cancers, bladder cancers (e.g., urinary bladder carcinomas),pancreatic cancers, liver cancers, kidney cancers (e.g., renal cellcarcinomas), squamous cell carcinomas, primary and metastatic CNS orbrain cancers (e.g., neuroblastomas, glioblastomas), melanomas such asmalignant melanomas, non-melanoma skin cancers, thyroid cancers (e.g.,medullary thyroid cancers (MTCs)), endometrial cancers, epithelialtumors bone cancers, and hematopoietic cancers, such as lymphomas (e.g.,T-cell lymphomas such as cutaneous T-cell lymphoma (CTCL), B-celllymphomas, small lymphocytic lymphoma, mangle cell lymphoma, anaplasticlarge cell lymphoma (ALCL), follicular lymphoma), leukemias (e.g.,chronic lymphocytic leukemia (CLL), hairy cell leukemia, acutelymphoblastic leukemia, myelocytic leukemia, acute myeloid ormyelogenous leukemia), multiple myeloma, Hodgkin's lymphoma, andnon-Hodgkin's lymphoma. Hence, in certain embodiments, a subject has oneor more of the above-described cancers.

Certain embodiments relate to methods for treating a cancer of thecentral nervous system (CNS), optionally the brain. In some embodiments,the cancer is a primary cancer of the CNS, such as a primary cancer ofthe brain. For instance, the methods can be for treating a glioma,meningioma, pituitary adenoma, vestibular schwannoma, primary CNSlymphoma, or primitive neuroectodermal tumor (medulloblastoma). In someembodiments, the glioma is an astrocytoma, oligodendroglioma,ependymoma, or a choroid plexus papilloma. In certain embodiments, theprimary CNS or brain cancer is glioblastoma multiforme, such as a giantcell gliobastoma or a gliosarcoma.

In particular embodiments, the cancer is a metastatic cancer of the CNS,for instance, a cancer that has metastasized to the brain. Examples ofsuch cancers include, without limitation, breast cancers, lung cancers,genitourinary tract cancers, gastrointestinal tract cancers (e.g.,colorectal cancers, pancreatic carcinomas), osteosarcomas, melanomas,head and neck cancers, prostate cancers (e.g., prostaticadenocarcinomas), and hematopoietic cancers such as lymphomas. Certainembodiments thus include methods for treating, inhibiting or preventingmetastasis of a cancer by administering to a patient a therapeuticallyeffective amount of a p97-antibody conjugate described herein (e.g., inan amount that, following administration, inhibits, prevents or delaysmetastasis of an antibody-resistant cancer in a statisticallysignificant manner, i.e., relative to an appropriate control as will beknown to those skilled in the art). In particular embodiments, thesubject has a cancer that has not yet metastasized to the centralnervous system, including one or more of the above-described cancers,among others known in the art.

In some aspects, the cancer or cancer cell is associated with expressionof at least one of Her2/neu, Her1/EGF receptor, Her3, A33 antigen, CD5,CD19, CD20, CD22, CD23 (IgE Receptor), C242 antigen, 5T4, IL-6, IL-13,vascular endothelial growth factor VEGF (e.g., VEGF-A) VEGFR-1, VEGFR-2,CD30, CD33, CD37, CD40, CD44, CD51, CD52, CD56, CD74, CD80, CD152,CD200, CD221, CCR4, HLA-DR, CTLA-4, NPC-1C, tenascin, vimentin,insulin-like growth factor 1 receptor (IGF-1R), alpha-fetoprotein,insulin-like growth factor 1 (IGF-1), carbonic anhydrase 9 (CA-IX),carcinoembryonic antigen (CEA), integrin α_(v)β₃, integrin α₅β₁, folatereceptor 1, transmembrane glycoprotein NMB, fibroblast activationprotein, alpha (FAP), glycoprotein 75, TAG-72, MUC1, MUC16 (or CA-125),phosphatidylserine, prostate-specific membrane antigen (PMSA), NR-LU-13antigen, TRAIL-R1, tumor necrosis factor receptor superfamily member 10b(TNFRSF10B or TRAIL-R2), SLAM family member 7 (SLAMF7), EGP40pancarcinoma antigen, B-cell activating factor (BAFF), platelet-derivedgrowth factor receptor, glycoprotein EpCAM (17-1A), Programmed Death-1,protein disulfide isomerase (PDI), Phosphatase of Regenerating Liver 3(PRL-3), prostatic acid phosphatase, Lewis-Y antigen, GD2 (adisialoganglioside expressed on tumors of neuroectodermal origin),glypican-3 (GPC3), or mesothelin. In specific aspects, the monoclonalantibody-portion of the p97-antibody conjugate specifically binds to oneor more of the foregoing cancer-associated antigens, or cancer antigens.

Particular aspects relate to the treatment of cancers associated withthe expression of Her2/neu. For example, one embodiment of the inventionprovides a method for treating, inhibiting or preventing a cancerincluding, but not limited to, Her2/neu-expressing breast cancer andmetastatic breast cancer, by administering to a patient atherapeutically effective amount of a herein disclosed conjugate. Anamount that, following administration, inhibits, prevents or delays theprogression and/or metastasis of a cancer in a statistically significantmanner (i.e., relative to an appropriate control as will be known tothose skilled in the art) is considered effective.

Another embodiment provides a method for treating, inhibiting orpreventing metastasis of a Her2/neu-expressing breast cancer byadministering to a patient a therapeutically effective amount of aherein disclosed conjugate (e.g., in an amount that, followingadministration, inhibits, prevents or delays metastasis of a cancer in astatistically significant manner, i.e., relative to an appropriatecontrol as will be known to those skilled in the art).

Particular aspects include the use of p97-trastuzumab for the treatmentof patients with metastatic breast cancer whose tumors overexpress theHer2 protein and have received one or more chemotherapy regiments fortheir metastatic disease. Some aspects include p97-trastuzumab incombination with paclitaxel for the treatment of patients withmetastatic breast cancer whose tumors overexpress the Her2 protein andwho have not received chemotherapy for their metastatic disease.

Specific aspects relate to the treatment of cancers associated with theexpression of Her1/EGFR. For instance, certain aspects include treatmentof a metastatic colorectal cancer or a head and neck cancer, where thep97-antibody conjugate specifically binds to Her1/EGFR and is an EGFRantagonist. In some aspects, the cancer is an EGFR-expressing metastaticcolorectal cancer, and is optionally KRAS wild-type. In particularaspects, the p97-antibody conjugate is administered to a subject withEGFR-expressing metastatic colorectal cancer after failure of bothirinotecan- and oxiplatin-based regimens. In some aspects, the subjectwith metastatic colorectal cancer is intolerant to irinotecan-basedregimens or is refractory to irinotecan-based chemotherapy. In otheraspects, the cancer is a locally or regionally advanced squamous cellcarcinoma of the head and neck, a recurrent locoregional disease ormetastatic squamous cell carcinoma of the head and neck, or a recurrentor metastatic squamous cell carcinoma of the head and neck progressingafter platinum-based therapy. In certain of these and relatedembodiments, the antibody portion of the p97-antibody conjugate iscetuximab, or an antigen-binding fragment thereof.

In specific embodiments, the p97-antibody conjugate comprises anantibody that specifically binds to human Her-2/neu, such astrastuzumab, and the subject optionally has breast cancer or metastaticbreast cancer, or a (metastatic) cancer of the CNS, as described herein.In other embodiments, the p97-antibody conjugate comprises an antibodythat specifically binds to GD2, such as 3F8, and the subject optionallyhas a neuroblastoma. In certain embodiments, the p97-antibody conjugatecomprises an antibody that specifically binds to CA-125, such asabagovomab, and the subject optionally has an ovarian cancer.

In particular embodiments, the p97-antibody conjugate comprises anantibody that specifically binds to EpCAM, such as adecatumumab, and thesubject optionally has a prostate cancer or breast cancer. In specificembodiments, the p97-antibody conjugate comprises an antibody thatspecifically binds to CD20, such as afutuzumab, and the subjectoptionally has a lymphoma. In specific embodiments, the p97-antibodyconjugate comprises an antibody that specifically binds to CD52, such asalemtuzumab, and the subject optionally has chronic lymphocytic leukemia(CLL) or CTCL.

In specific embodiments, the p97-antibody conjugate comprises anantibody that specifically binds to VEGF-R2, such as alacizumab (pegol).In specific embodiments, the p97-antibody conjugate comprises anantibody that specifically binds to HLA-DR, such as apolizumab, and thesubject optionally has a hematological cancer. In specific embodiments,the p97-antibody conjugate comprises an antibody that specifically bindsto BAFF, such as belimumab, and the subject optionally has ahematopoietic cancer such as Non-Hodgkin's lymphoma.

In specific embodiments, the p97-antibody conjugate comprises anantibody that specifically binds to VEGF-A, such as bevacizumab, and thesubject optionally has a metastatic cancer or colorectal cancer. Inspecific embodiments, the p97-antibody conjugate comprises an antibodythat specifically binds to CD44, such as bivatuzumab (mertansine), andthe subject optionally has a squamous cell carcinoma. In specificembodiments, the p97-antibody conjugate comprises an antibody thatspecifically binds to CD30, such as brentuximab vedotin, and the subjectoptionally has a hematological cancer such as anaplastic large celllymphoma (ALCL) or Hodgkin's lymphoma.

In specific embodiments, the p97-antibody conjugate comprises anantibody that specifically binds to mucin, such as cantuzumab(mertansine), and the subject optionally has a colorectal cancer. Inspecific embodiments, the p97-antibody conjugate comprises an antibodythat specifically binds to PSMA, such as capromab, and the subjectoptionally has a prostate cancer. In specific embodiments, thep97-antibody conjugate comprises an antibody that specifically binds toEpCAM and optionally CD3, such as cetuximab, and the subject optionallyhas a (metastatic) colorectal cancer or a head and neck cancer.

In certain embodiments, the p97-antibody conjugate comprises an antibodythat specifically binds to EpCAM, such as citatuzumab (bogatox), and thesubject optionally has a solid tumor such as ovarian cancer. Inparticular embodiments, the p97-antibody conjugate comprises an antibodythat specifically binds to the IGF-1 receptor, such as cixutumumab, andthe subject optionally has a solid tumor. In specific embodiments, thep97-antibody conjugate comprises an antibody that specifically binds toMUC1, such as clivatuzumab (tetraxetan), and the subject optionally hasa pancreatic cancer.

In specific embodiments, the p97-antibody conjugate comprises anantibody that specifically binds to CD40, such as dacetuzumab, and thesubject optionally has a hematologic cancer. In some embodiments, thep97-antibody conjugate comprises an antibody that specifically binds toGD3 ganglioside, such as ecromeximab, and the subject optionally has amalignant melanoma. In specific embodiments, the p97-antibody conjugatecomprises an antibody that specifically binds to EpCAM, such asedrecolomab, and the subject optionally has a colorectal carcinoma.

In specific embodiments, the p97-antibody conjugate comprises anantibody that specifically binds to SLAMF7, such as elotuzumab, and thesubject optionally has a multiple myeloma. In specific embodiments, thep97-antibody conjugate comprises an antibody that specifically binds tointegrin α_(v)β₃, such as etaracizumab, and the subject optionally has amelanoma, prostate cancer, ovarian cancer, or other solid tumor. Incertain embodiments, the p97-antibody conjugate comprises an antibodythat specifically binds to folate receptor 1, such as farletuzumab, andthe subject optionally has an ovarian cancer.

In specific embodiments, the p97-antibody conjugate comprises anantibody that specifically binds to the IGF-1 receptor, such asfigitumumab, and the subject optionally has adrenocortical carcinoma ornon-small cell lung carcinoma. In specific embodiments, the p97-antibodyconjugate comprises an antibody that specifically binds to glycoprotein75, such as flanvotumab, and the subject optionally has a melanoma. Inspecific embodiments, the p97-antibody conjugate comprises an antibodythat specifically binds to CD80, such as galiximab, and the subjectoptionally has a B-cell lymphoma.

In particular embodiments, the p97-antibody conjugate comprises anantibody that specifically binds to CD33, such as gemtuzumab(ozogamicin), and the subject optionally has a myelogenous leukemia. Inspecific embodiments, the p97-antibody conjugate comprises an antibodythat specifically binds to CA-IX, such as girentuximab, and the subjectoptionally has a renal cell carcinoma. In further embodiments, thep97-antibody conjugate comprises an antibody that specifically binds toGPMNB, such as glembatumumab (vedotin), and the subject optionally has amelanoma or breast cancer.

In specific embodiments, the p97-antibody conjugate comprises anantibody that specifically binds to CD20, such as ibritumomab tiuxetan,and the subject optionally has a hematopoietic cancer such asnon-Hodgkin's lymphoma. In some embodiments, the p97-antibody conjugatecomprises an antibody that specifically binds to CTLA-4, such asipilimumab (MDX-101), and the subject optionally has a solid tumor suchas a melanoma. In specific embodiments, the p97-antibody conjugatecomprises an antibody that specifically binds to CD51, such asintetumumab, and the subject optionally has a solid tumor such asprostate cancer or melanoma.

In particular embodiments, the p97-antibody conjugate comprises anantibody that specifically binds to CD30, such as iratumumab, and thesubject optionally has a hematopoietic cancer such as Hodgkin'slymphoma. In specific embodiments, the p97-antibody conjugate comprisesan antibody that specifically binds to CEA, such as labetuzumab, and thesubject optionally has a colorectal cancer. In specific embodiments, thep97-antibody conjugate comprises an antibody that specifically binds toCD40, such as lucatumumab, and the subject optionally has ahemotopoietic cancer such as multiple myeloma, non-Hodgkin's lymphoma,or Hodgkin's lymphoma.

In certain embodiments, the p97-antibody conjugate comprises an antibodythat specifically binds to CD23, such as lumiliximab, and the subjectoptionally has CLL. In specific embodiments, the p97-antibody conjugatecomprises an antibody that specifically binds to EGFR, such asmatuzumab, and the subject optionally has a colorectal, lung, or stomachcancer. In specific embodiments, the p97-antibody conjugate comprises anantibody that specifically binds to CD74, such as milatuzumab, and thesubject optionally has a hemotological cancer such as multiple myeloma.

In some embodiments, the p97-antibody conjugate comprises an antibodythat specifically binds to GD3 ganglioside, such as mitumomab, and thesubject optionally has a small cell lung carcinoma. In specificembodiments, the p97-antibody conjugate comprises an antibody thatspecifically binds to 5T4, such as naptumomab (estafenatox), and thesubject optionally has a non-small cell lung carcinoma or renal cellcarcinoma. In specific embodiments, the p97-antibody conjugate comprisesan antibody that specifically binds to EGFR, such as necitumumab, andthe subject optionally has a non-small cell lung carcinoma.

In particular embodiments, the p97-antibody conjugate comprises anantibody that specifically binds to EGFR, such as nimotuzumab, and thesubject optionally has a squamous cell carcinoma, head and neck cancer,nasopharyngeal cancer, or glioma. In specific embodiments, thep97-antibody conjugate comprises an antibody that specifically binds toCD20, such as ofatumumab, and the subject optionally has a hematopoieticcancer such as CLL. In specific embodiments, the p97-antibody conjugatecomprises an antibody that specifically binds to CA-125, such asoregovomab, and the subject optionally has an ovarian cancer.

In specific embodiments, the p97-antibody conjugate comprises anantibody that specifically binds to EGFR, such as panitumumab, and thesubject optionally has a colorectal cancer. In some embodiments, thep97-antibody conjugate comprises an antibody that specifically binds tovimentin, such as pritumumab, and the subject optionally has a braincancer. In specific embodiments, the p97-antibody conjugate comprises anantibody that specifically binds to CD20, such as rituximab, and thesubject optionally has a hematopoietic cancer such as a lymphoma orleukemia.

In certain embodiments, the p97-antibody conjugate comprises an antibodythat specifically binds to CD20, such as tositumomab, and the subjectoptionally has a lymphoma such as follicular lymphoma. In specificembodiments, the p97-antibody conjugate comprises an antibody thatspecifically binds to GD2, such as TRBS07, and the subject optionallyhas a melanoma. In specific embodiments, the p97-antibody conjugatecomprises an antibody that specifically binds to integrin α₅β₁, such asvolociximab, and the subject optionally has a solid tumor.

In particular embodiments, the p97-antibody conjugate comprises anantibody that specifically binds to tumor antigen CTAA16.88, such asvotumumab, and the subject optionally has a colorectal tumor. Inspecific embodiments, the p97-antibody conjugate comprises an antibodythat specifically binds to EGFR, such as zalutumumab, and the subjectoptionally has a squamous cell carcinoma of the head and neck.

As noted above, the use of p97-antibody conjugates for treating cancerscan be combined with other therapeutic modalities. For example, acomposition comprising a p97-antibody conjugate can be administered to asubject before, during, or after other therapeutic interventions,including symptomatic care, radiotherapy, surgery, transplantation,immunotherapy, hormone therapy, photodynamic therapy, chemotherapy,antibiotic therapy, or any combination thereof. Symptomatic careincludes administration of corticosteroids, to reduce cerebral edema,headaches, cognitive dysfunction, and emesis, and administration ofanti-convulsants, to reduce seizures. Radiotherapy includes whole-brainirradiation, fractionated radiotherapy, and radiosurgery, such asstereotactic radiosurgery, which can be further combined withtraditional surgery.

In specific combination therapies, the antibody portion of thep97-antibody conjugate comprises cetuximab, and the p97-cetuximabconjugate is used for treating a subject with locally or regionallyadvanced squamous cell carcinoma of the head and neck in combinationwith radiation therapy. In other aspects, the p97-cetuximab conjugate isused for treating a subject with recurrent locoregional disease ormetastatic squamous cell carcinoma of the head and neck in combinationwith platinum-based therapy with 5-fluorouracil (5-FU). In some aspects,the p97-cetuximab conjugate is used in combination with irinotecan fortreating a subject with EGFR-expressing colorectal cancer and that isrefractory to irinotecan-based chemotherapy.

Methods for identifying subjects with one or more of the diseases orconditions described herein are known in the art.

For in vivo use for the treatment of human disease, the conjugatesdescribed herein are generally incorporated into a pharmaceuticalcomposition prior to administration. A pharmaceutical compositioncomprises one or more of the conjugates described herein in combinationwith a physiologically acceptable carrier or excipient as describedelsewhere herein. To prepare a pharmaceutical composition, an effectiveamount of one or more of the compounds is mixed with any pharmaceuticalcarrier(s) or excipient known to those skilled in the art to be suitablefor the particular mode of administration. A pharmaceutical carrier maybe liquid, semi-liquid or solid. Solutions or suspensions used forparenteral, intradermal, subcutaneous or topical application mayinclude, for example, a sterile diluent (such as water), salinesolution, fixed oil, polyethylene glycol, glycerin, propylene glycol orother synthetic solvent; antimicrobial agents (such as benzyl alcoholand methyl parabens); antioxidants (such as ascorbic acid and sodiumbisulfite) and chelating agents (such as ethylenediaminetetraacetic acid(EDTA)); buffers (such as acetates, citrates and phosphates). Ifadministered intravenously, suitable carriers include physiologicalsaline or phosphate buffered saline (PBS), and solutions containingthickening and solubilizing agents, such as glucose, polyethyleneglycol, polypropylene glycol and mixtures thereof.

The compositions comprising conjugates as described herein may beprepared with carriers that protect the conjugates against rapidelimination from the body, such as time release formulations orcoatings. Such carriers include controlled release formulations, suchas, but not limited to, implants and microencapsulated delivery systems,and biodegradable, biocompatible polymers, such as ethylene vinylacetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylacticacid and others known to those of ordinary skill in the art.

The following Examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1 Conjugation of P97 to Anti-Her2/Neu Antibodies

Trastuzumab, a humanized monoclonal antibody specific for the Her2/neuprotein and used clinically in the treatment of HER2+ breast cancer, waschemically linked to a p97 delivery vector (Transcend; BiOasis), asdescribed below.

Initial Preparation of Antibody

1. Approximately 100 mg (formulated weight including excipients, etc.)of “BTA” antibody (Roche), which specifically binds to the humanHer2/neu protein, was dissolved in 1.5 ml of deionized water andbuffer-exchanged into 0.1 M potassium phosphate buffer pH 7.5 on asingle PD10 column (GE 170851-01), yielding 3.0 ml of an antibodysolution at 18.10 mg/ml as indicated by UV-visible spectrophotometry at280 nm, and assuming an absorbance of 1.40 at this wavelength for a 1mg/ml solution of antibody (Antibody A).

2. Approximately 100 mg (formulated weight including excipients, etc.)of “BTA” antibody (Roche) was dissolved in 4.0 ml of deionised water andbuffer-exchanged into 0.1 M potassium phosphate buffer pH 7.5 on threePD10 columns (GE 170851-01), yielding 8.1 ml of an antibody solution at6.50 mg/ml as indicated by UV-visible spectrophotometry at 280 nm, andassuming an absorbance of 1.40 at this wavelength for a 1 mg/ml solutionof antibody (Antibody B).

Cy5.5 Labeling of Antibody

3. To 53.4 mg (2.95 ml) of Antibody B was added 1.89 mg (189 ul) of a10.0 mg/ml solution of Cy5.5 NHS ester (Lumiprobe 24020) in DMSO,equivalent to a 7:1 Cy5.5: antibody excess.

4. The dye-antibody reaction was allowed to continue for 60 minutes at20° C.

5. The crude antibody-dye conjugate was purified using size exclusionchromatography on two PD10 columns, using 0.1 M potassium phosphate pH7.5 as eluent, to remove low-molecular weight by-products. This yieldeda solution of dye-labelled antibody with an antibody concentration of9.45 mg/ml and with an apparent incorporation of 2.01 dye molecules perantibody molecule as indicated by UV-visible spectrophotometry at 280 nmand 673 nm, and assuming an absorbance of 1.40 at this wavelength for a1 mg/ml solution of antibody and a molar extinction coefficient of133,000 M⁻¹ cm⁻¹ at 673 nm for Cy5.5 (Dye-Labelled Antibody B)

Incorporation of Maleimides into Unlabelled Antibody

6. To 40 mg (6.15 ml) of Antibody A was added 0.31 mg (62 ul) of a 5.0mg/ml solution of 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC,Thermo 22360) in DMSO, equivalent to an SMCC: antibody excess of 3.5:1.

7. The antibody activation reaction was allowed to continue for 60minutes at 20° C.

8. The crude maleimide-activated unlabelled antibody was purified usingsize exclusion chromatography on four PD10 columns to removelow-molecular weight by-products, using 50 mM potassium phosphate, 150mM sodium chloride, 5 mM EDTA buffer pH 7.0 buffer as eluent. Thisyielded a solution of maleimide-activated, unlabelled antibody with anantibody concentration of 3.34 mg/ml assuming an absorbance of 1.40 atthis wavelength for a 1 mg/ml solution of antibody. (Maleimide-ActivatedUnlabelled Antibody A1)

9. To 5 mg (769 ul) of Antibody A was added 0.17 mg (33 ul) of a 5.0mg/ml solution of 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC,Thermo 22360) in DMSO, equivalent to an SMCC: antibody excess of 15:1.

10. The antibody activation reaction was allowed to continue for 60minutes at 20° C.

11. The crude maleimide-activated, unlabelled antibody was purifiedusing size exclusion chromatography on a single PD10 column to removelow-molecular weight by-products, using 50 mM potassium phosphate, 150mM sodium chloride, 5 mM EDTA buffer pH 7.0 buffer as eluent. Thisyielded a solution of maleimide-activated, unlabelled antibody with anantibody concentration of 2.36 mg/ml assuming an absorbance of 1.40 atthis wavelength for a 1 mg/ml solution of antibody. (Maleimide-ActivatedUnlabelled Antibody A2)

Incorporation of Maleimides into Cy5.5-Labelled Antibody

12. To 40 mg (4.23 ml) of Dye-Labelled Antibody B was added 0.45 mg (89ul) of a 5.0 mg/ml solution of4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC, Thermo 22360) inDMSO, equivalent to an SMCC: antibody excess of 5:1.

13. The antibody activation reaction was allowed to continue for 60minutes at 20° C.

14. The crude maleimide-activated, dye-labelled antibody was purifiedusing size exclusion chromatography on three PD10 columns to removelow-molecular weight by-products, using 50 mM potassium phosphate, 150mM sodium chloride, 5 mM EDTA buffer pH 7.0 buffer as eluent. Thisyielded a solution of maleimide-activated, dye-labelled antibody with anantibody concentration of 4.46 mg/ml and with an apparent incorporationof 1.05 dye molecules per antibody molecule as indicated by UV-visiblespectrophotometry at 280 nm and 673 nm, and assuming an absorbance of1.40 at this wavelength for a 1 mg/ml solution of antibody and a molarextinction coefficient of 133,000 M⁻¹ cm⁻¹ at 673 nm for Cy5.5.(Maleimide-Activated Dye-Labelled Antibody B1)

15. To 5 mg (529 ul) of Dye-Labelled Antibody B was added 0.20 mg (40ul) of a 5.0 mg/ml solution of4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC, Thermo 22360) inDMSO, equivalent to an 18:1 SMCC: antibody excess.

16. The antibody activation reaction was allowed to continue for 60minutes at 20° C.

17. The crude maleimide-activated, dye-labelled antibody was purifiedusing size exclusion chromatography on a single PD10 column to removelow-molecular weight by-products, using 50 mM potassium phosphate, 150mM sodium chloride, 5 mM EDTA buffer pH 7.0 buffer as eluent. Thisyielded a solution of maleimide-activated, dye-labelled antibody with anantibody concentration of 1.85 mg/ml and with an apparent incorporationof 0.66 dye molecules per antibody molecule as indicated by UV-visiblespectrophotometry at 280 nm and 673 nm, and assuming an absorbance of1.40 at this wavelength for a 1 mg/ml solution of antibody and a molarextinction coefficient of 133,000 M⁻¹ cm⁻¹ at 673 nm for Cy5.5.(Maleimide-Activated Dye-Labelled Antibody B2)

Initial Preparation of p97

18. In parallel with steps 1-17, approximately 70 mg of p97 (BiOasis)was buffer-exchanged into 0.1 M potassium phosphate buffer pH 7.5 onthree PD10 columns, yielding 11.2 ml of a p97 solution at 6.37 mg/ml asindicated by UV-visible spectrophotometry at 280 nm, and assuming anabsorbance of 1.19 at this wavelength for a 1 mg/ml solution of p97.

Thiolation of p97

19. To 54 mg (8.48 ml) of the buffer-exchanged p97 was added 0.41 mg (82ul) of a 5.0 mg/ml solution of S-acetylthioacetic acid, succinimidylester (SATA, Thermo 26102) in DMSO, equivalent to a SATA: p97 ratio of3.2:1.

20. The S-acetylthiolation reaction was allowed to proceed for 55minutes at 20° C.

21. 848 ul of an aqueous solution of 0.05M EDTA disodium salt and 2.5Mhydroxylamine hydrochloride, pH 7.0, was added to deprotect the thiols,the deprotection reaction being allowed to proceed for 17 minutes at 20°C.

22. The crude thiolated p97 was purified using size exclusionchromatography on five PD10 columns to remove low-molecular weightby-products, using 50 mM potassium phosphate, 150 mM sodium chloride, 5mM EDTA buffer pH 7.0 buffer as eluent. This yielded a solution ofthiolated p97 with a p97 concentration of 3.65 mg/ml as indicated byUV-visible spectrophotometry at 280 nm, and assuming an absorbance of1.19 at this wavelength for a 1 mg/ml solution of p97. A sample wasassayed for thiol content using Ellman's Reagent, indicating anincorporation of 0.9 thiol groups per p97 molecule. (Thiolated p97 C1)

23. To 8 mg (1.26 ml) of the buffer-exchanged p97 was added 0.29 mg (57ul) of a 5.0 mg/ml solution of S-acetylthioacetic acid, succinimidylester (SATA, Thermo 26102) in DMSO, equivalent to a SATA: p97 ratio of15:1.

24. The S-acetylthiolation reaction was allowed to proceed for 55minutes at 20° C.

25. 126 ul of an aqueous solution of 0.05M EDTA disodium salt and 2.5Mhydroxylamine hydrochloride, pH 7.0, was added to deprotect the thiols,the deprotection reaction being allowed to proceed for 17 minutes at 20°C.

26. The crude thiolated p97 was purified using size exclusionchromatography on a single PD10 column to remove low-molecular weightby-products, using 50 mM potassium phosphate, 150 mM sodium chloride, 5mM EDTA buffer pH 7.0 buffer as eluent. This yielded a solution ofthiolated p97 with a p97 concentration of 2.83 mg/ml as indicated byUV-visible spectrophotometry at 280 nm, and assuming an absorbance of1.19 at this wavelength for a 1 mg/ml solution of p97. A sample wasassayed for thiol content using Ellman's Reagent, indicating anincorporation of 3.8 thiol groups per p97 molecule. (Thiolated p97 C2)

p97-Antibody Conjugations

27. (BOA2/1) 5.0 mg (1.50 ml) of Maleimide-Activated Unlabelled AntibodyA1 and 12.93 mg (3.55 ml) of Thiolated p97 C1, equivalent to a p97:antibody ratio of 4.0:1, were allowed to react together for 18 hours at20° C.

28. (BOA2/2) 5.0 mg (1.12 ml) of Maleimide-Activated Dye-LabelledAntibody B1 and 12.93 mg (3.55 ml) of Thiolated p97 C1, equivalent to ap97: antibody ratio of 4.0:1, were allowed to react together for 18hours at 20° C.

29. (BOA2/3) 2.5 mg (1.06 ml) of Maleimide-Activated Unlabelled AntibodyA2 and 12.93 mg (3.55 ml) of Thiolated p97 C1, equivalent to a p97:antibody ratio of 8.0:1, were allowed to react together for 18 hours at20° C.

30. (BOA2/4) 2.5 mg (1.35 ml) of Maleimide-Activated Dye-LabelledAntibody 82 and 12.93 mg (3.55 ml) of Thiolated p97 C1, equivalent to ap97: antibody ratio of 8.0:1, were allowed to react together for 18hours at 20° C.

31. (BOA2/5) 23.0 mg (6.89 ml) of Maleimide-Activated UnlabelledAntibody A1 and 2.47 mg (0.87 ml) of Thiolated p97 C2, equivalent to ap97: antibody ratio of 1:6.0, were allowed to react together for 18hours at 20° C.

32. (BOA2/6) 23.0 mg (5.16 ml) of Maleimide-Activated Dye-LabelledAntibody 81 and 2.47 mg (0.87 ml) of Thiolated p97 C2, equivalent to ap97: antibody ratio of 1:6.0, were allowed to react together for 18hours at 20° C. 33. The six crude antibody-p97 and antibody-Cy5.5-p97conjugates from steps 27-32 were concentrated to 1-1.5 ml using Vivaspin6 (30 kDa cut-off) spin filters and purified by high-resolution sizeexclusion chromatography, using a 1.6×36 cm Superdex 200PG column at 2.0ml/min with 50 mM potassium phosphate buffer+150 mM sodium chloride, pH6.7, as the eluent.

34. This yielded conjugates with approximate p97: antibody ratios shownbelow, as indicated by UV-spectrophotometric and size-exclusionchromatography data.

TABLE 2 Conjugate P97:antibody ratio (BOA2/1) 1.3:1   (BOA2/2) 1.5:1  (BOA2/3) 3:1 (BOA2/4) 3:1 (BOA2/5)   1:2.5 (BOA2/6)   1:2.5

Example 2 P97-Her2/Neu Antibody Conjugates Enhance Cell Death of BreastCancer Cells

This example demonstrates that p97-Her2/neu antibody conjugates of theinvention have enhanced activity against breast cancer cells comparedwith anti-Her2/neu antibodies that are not conjugated with p97.

The compounds noted below were tested against the breast cancer celllines MCF-7 vector, MCF-7/Her2, BT474, and SKBr3, as further describedbelow. Vehicle was PBS, pH 6.7.

TABLE 3 Compound # Molecular Weight (g/mol) Trastuzumab 145,531.50BOA2/1 250,000.00 BOA2/3 760,000.00 BOA2/5 530,000.00 hMTF (p97)76,000.00

Materials & Methods Cell Lines

The MCF-7 vector (human breast cancer) cell line was maintained underthe following conditions: RPMI 1640 media, 2 mM L-glutamine, 10% FBS,500 μg/mL G418. The MCF-7 HER2 (human breast cancer, transfected) cellline was maintained under the following conditions: RPMI 1640 media, 2mM L-glutamine, 10% FBS, 500 μg/mL G418. The SKBR3 (human breast cancer)cell line was maintained under the following conditions: McCoys 5Amedia, 1.5 mM L-glutamine, 10% FBS. The BT474 (human breast cancer) cellline was maintained under the following conditions: DMEM media, 2 mML-glutamine, 10% FBS

Cell Growth Optimization

A cell growth optimization study is performed prior to the drugscreening study. The objective of the cell growth study is to determinethe optimal seeding density for each cell line to ensure that 95-100%confluency is reached after 96 hour incubation (in both low and highserum conditions) and to determine the optimal staining conditions forHoechst 33342 (H33342). The following cell densities are tested: 500,1000, 1500, 2000 2500 and 3000 cells/well in 50 μL in normal serumconditions (Table 4) and 2000, 4000, 6000, 8000, 10000 and 12000cells/well in 50 μL in low (1%) serum conditions (Table 5). Cells arediluted to different concentration in a 2 mL 96-well deep block based onthe following tables:

TABLE 4 Normal Serum Conditions Volume of stock Volume of cell cellsolution (mL) Final Cell Number of culture media for (stock =Concentration Cells (50 μL) dilution (mL) 60,000 cells/mL) (cells/mL)3,000 0.000 2.000 60000 2,500 0.333 1.667 50000 2,000 0.667 1.333 400001,500 1.000 1.000 30000 1,000 1.333 0.667 20000 500 1.667 0.333 10000

TABLE 5 Low Serum Conditions Volume of stock Volume of cell cellsolution (mL) Final Cell Number of culture media for (*stock =Concentration10 Cells (50 μL) dilution (mL) 240,000 cells/mL) (cells/mL)12,000 0.000 2.000 240,000 10,000 0.333 1.667 200,000 8,000 0.667 1.333160,000 6000 1.000 1.000 120,000 4,000 1.333 0.667 80,000 2,000 1.6670.333 40,000*Take 60,000/25 mL stock solution of cells and spin down and add 6.5 mLmedia

Plating is done with the Hydra by transferring from the 96 well deepblock to 4 quadrant wells in corresponding Grenier Bio or with amulti-channel pipette. Each well of the 384-well plate has 50 μL addedand this is repeated. After 24 hours, the media is aspirated andreplaced with media containing normal serum or no serum. During theaspiration step, approximately 45 μL of media is removed and replacedwith new media. Staining is performed daily on 1 plate which will beimaged. Images are compared over the time course for each cell line todetermine optimal seeding density.

Hoechst 33342 Staining Optimization

Optimization of the Hoechst 33342 is important to give the strongestsignal-to-noise ratio without precipitating the dye or killing thecells. Hoechst 33342 is a cell permeable dye which labels the cellnuclei allowing for efficient image-based segmentation and counting.Four different concentrations of Hoechst 33342 are tested (0.5, 1, 5, 10μM) for effectiveness. EthD1 is a membrane impermeable nucleic acid dyethat binds to DNA in the nucleus of cells with compromised cellmembranes (i.e. dead cells). EthD1 is used at a final concentration of 1μM. The two stains provide a systematic way of identifying live/deadcells for efficient cell counting. Using 1 plate, Hoechst 33342 arediluted in cell culture media to the working concentrations indicated inthe following table:

TABLE 6 Final Intermediate final concentration concentration stock dyediluting media volume H33342 (μM) H33342 (μM) (1 mM) (μL) (μL) 0.5 4 1.2298.8 300 1 8 2.4 297.6 300 5 40 12 288.0 300 10 80 24 276.0 30010 μL of the staining solution is added to each well and incubated for30 minutes at 37° C., 5% CO₂ to allow for uptake of the dye. Imageoptimization is achieved on the InCell Analyzer 1000 machine.

Drug Screening Protocol: Day 0: Cell Plating-384 Well Plates:

Cells are harvested and plated in two 384 well Grenier Bio One TCtreated μClear 384 plates based on the optimal cell numbers in the cellgrowth optimization study. 50 μL from the stock cell solution is addedto the assay plate according to the Assay Plate Layout (120 wells/cellline)

Day 1: Drug Treatment:

Serial dilutions of Test Articles are performed in 96 well plates.Following dilutions of Test and Control Articles, 20 μL/well×2 wells perdrug concentration are added according to the Assay Plate Layout shownin FIG. 13.

The Table below shows illustrative dilutions of stocks to give desiredworking solutions.

TABLE 7 Dilutions of stocks to give a working solution. Doses based onClin Cancer Res 2004; 10: 2512-2524. Published online Apr. 8, 2004. TestStock Conc Stock vol Total Vol Final Molar Final Conc Material(moles/uL) (uL) Media (uL) (uL) Conc (ug/ml) Herceptin 3.96E−11 17 9831000 6.87E−07 100 BOA2/1 2.72E−12 253 747 1000 6.87E−07 172 BOA2/31.12E−12 614 386 1000 6.87E−07 522 BOA2/5 1.72E−12 400 600 1000 6.87E−07364 p97 1.32E−10 5 995 1000 6.87E−07 50 *These are dilutions to give 1mL of material. A 1 in 10 dilution of this gives 10 μg/mL equivalent.

The dilution scheme used in the plate is that outlined above in“Exemplary Drug Dilution Reservoir.”

TABLE 8 Final Conc (ug/ml) Dilution Final Molar Conc (M) herceptinequivalents neat 6.87E−07 100 1:10 6.87E−08 10 1:100 6.87E−09 1 1:10006.87E−10 0.1 1:3.3 2.29E−07 30 1:33 2.29E−08 3 1:333 2.29E−09 0.3

Day 2-3: Incubation:

Cells are incubated with drug for 72 hours at 37° C., 5% CO₂, humidifiedincubator.

Day 4: Imaging:

Plates are stained with Hoechst 33342 (total cells) based on stainingoptimization study, and Ethidium Homodimer (dead cells) to determineviable cell counts. Plates are imaged with GE InCell 1000 CellularImaging and Analysis System.

Results

Using procedures substantially as described above, various conjugates ofthe invention were tested for activity against breast cancer cell linesin comparison with an unconjugated anti-Her2/neu antibody. Results ofcell viability assays are summarized in FIGS. 1-4. Surprisingly, in someHER2+ breast cancer cell lines, p97-antibody conjugates demonstrated asignificant improvement in cancer killing activity compared totrastuzumab alone. For example, for the BT474 cell line (FIG. 1),conjugates BOA2/1 and BOA2/5 showed a profound effect on cell death at229 nM. The effect of these conjugates on cell viability was much morepronounced than the effect observed for cells treated withtrastuzumab-alone (BOA2/8).

In addition, the results of other experiments confirmed that trastuzumabdoes not enter human brain endothelial (HBE) cells in culture nor doesit cross the intact blood brain barrier in animals. However,p97-antibody conjugates showed a marked transport into HBE cells,suggesting that the conjugates have the potential to cross theblood-brain barrier and enter brain tissue.

In light of these findings, it is clear that the conjugates of thepresent invention can improve the therapeutic potential of anti-Her2/neuantibodies by improving the activity of the antibodies and/or allowingthe antibodies to access Her2/neu-expressing metastatic cancer cells inthe CNS.

Example 3 Distribution of p97-Trastuzumab Conjugates in Brain Tissue

Experiments were performed to evaluate distribution of p97-trastuzumab(Herceptin®) conjugates in brain tissue compartments. First, thefollowing rhodamine-labeled proteins were injected intravenously intomice about 23 grams in size: 100 μg p97-rhodamine at about 4.35 mg/kg;195 μg p97-rhodamine at about 8.47 mg/kg; 375 μg p97-BTA-rhodamine atabout 16.3 mg/kg; and rhodamine alone.

For labeled proteins, three 20 μm sections and three 50 μm sections werecollected at 2 hours post-IV injection, and two 20 μm sections and two50 μm sections were collected at 24 hours post-IV injection. Forrhodamine alone, four 20 μm sections and two 50 μm sections werecollected at 2 hours post-IV injection. These sections were sent toiCapture Imaging Facility for confocal analysis.

The number of voxels were measured in the 20 micron sections of thevascular compartment (capillaries) and the brain parenchyma (all tissueexcept the vascular compartment). A voxel is a three dimensional pixeltaken from confocal imaging of a tissue section. The number offluorescent voxels in the brain parenchyma was divided by the totalnumber of voxels to give the volume fraction of a given conjugate in theparenchyma. The number of fluorescent voxels in the brain capillarieswas divided by the total number of voxels to give the volume fraction ofa conjugate in the vasculature (capillaries).

The results are shown in FIGS. 5A-5D. In these figures “MTf” is p97 and“BTA” is trastuzumab. FIG. 5A shows the distribution of p97-trastuzumab(BT2111; MTf-BTA) in brain tissue when normalized to injected dose, andFIG. 5B shows the same when normalized to fluorescence. Here, most ofthe fluorescent proteins are found in the capillaries, but relative top97 alone or trastuzumab alone, the p97-trastuzumab conjugateselectively distributes to the parenchyma, especially at the 2 hourtime-point. This result is further illustrated in FIG. 5C, which showssignificantly increased fluorescence localized to the brain parenchymafor p97-trastuzumab conjugate, and FIG. 5D, which shows that theparenchymal levels of the p97-trastuzumab conjugate are 12-fold greaterthan trastuzumab alone and 4-fold greater than p97 alone.

These findings suggest that the combination of p97 and trastuzumab as aprotein conjugate synergistically increases delivery to parenchymalbrain tissues across the blood brain barrier, relative to the deliveryof each protein alone.

Example 4 Distribution of p97-Trastuzumab Conjugates in Brain Metastases

Experiments were performed to evaluate the distribution of ²⁵I-labeledp97-trastuzumab conjugate in normal brain tissue and brain metastases,relative to trastuzumab alone. The relative distribution in systemictissues was also examined.

Intravenous drug administration of radio-labeled p97, trastuzumab, andp97-trastuzumab was performed on mice having experimental brainmetastases of breast cancer. Specifically, these tumor experimentsutilized immune compromised NuNu mice (Charles River Labs) that wereimplanted via intracardiac injection with eGFP-expressing MDA-MB-231 BRHigh Her-2 human breast cancer cells. The tumor cells were allowed toimplant in the brain and form brain metastases over a period of about3-6 weeks.

Test drugs were administered once the animals start exhibiting symptomsof tumor growth. Monitoring for tissue distribution then was performedby radioactive, fluorescent, and quantitative autoradiography analysis.These experiments were performed using ¹²⁵I-labeled p97, trastuzumab,and p97-trastuzumab proteins at a purity of >99%, as measured by HPLC.

Uptake of radio-labeled proteins was examined at two, eight, and 24 hourtime points. Texas red-dextran was also administered (i.v.) at about 10min-2 hr prior to euthanasia to measure blood-tumor barrier passivepermeability. Brains were perfused with 2.7% albumin and iodocyaninegreen for about 30-60 seconds after euthanasia to map the distributionof blood vessels within the brain and brain metastases, and to removeintravascular labeled protein. Brains were snap-frozen immediatelypost-perfusion washout and were cut into 20 μm coronal sections using acryostat. The sections were analyzed for green, red, and near infraredfluorescence, to respectively map brain distribution of tumor cells,quantitate blood-tumor barrier permeability, and localize vasculaturewithin tumors. In matching tissue sections, the distribution of¹²⁵I-protein in brain was measured by phosphorescence imaging along withradioactive standards. After analysis, tissue sections were stained toconfirm tumor cell distribution. Dissection was also performed tomeasure and compare the level/distribution of ¹²⁵I-protein (dpm/g) inother tissues, including the liver, kidneys, lung, heart, spleen,muscle, and fat.

Image analysis was used to determine the level of ¹²⁵I-labeled proteinin selected brain metastases and surrounding normal brain, and to obtainautoradiographic images expressed in units of nCi/g tissue or ngprotein/g tissue. The uptake of labeled proteins in brain metastasis wasanalyzed in relation to metastasis size, blood-tumor barrierpermeability, and time of circulation. Calculations were also performedto measure the percentage dose/g or ml of intact drug to brain, brainmetastasis, blood and other tissues.

The results are shown in FIGS. 6-10. FIGS. 6 and 7 show the results for¹²⁵I-labeled trastuzumab, and FIGS. 8-10 show the results for¹²⁵I-labeled p97-trastuzumab conjugate.

FIGS. 6A-6F show the distribution of ¹²⁵I-labeled trastuzumab in themouse brain at 24 hours post-intravenous administration. FIG. 6A showsbrain metastases of heterogenous size within the regions outlined inred, and FIG. 6B shows Texas Red-Dextran staining of the metastases.FIG. 6C shows an autoradiogram of ¹²⁵I-labeled trastuzumab, andindicates the fold increase in antibody relative to the surroundingnormal brain tissue. Here, the ¹²⁵I-labeled trastuzumab is at the limitof detection. As shown in FIG. 6F, the K_(in) values for trastuzumabalone are about 1.46×10⁻⁷ mL/sec/g in normal brain tissue and about3.8×10⁻⁷ mL/sec/g in brain metastases, relatively low K_(in) values fora protein and about ˜1000 lower than the K_(in) values forp97-trastuzumab conjugate.

FIGS. 7A-7D show the distribution of ¹²⁵I-labeled trastuzumab in themouse brain and other tissues at 24 hours post-intravenousadministration. FIG. 7A shows brain metastases of heterogenous sizewithin the regions outlined in red, and FIG. 7B shows Texas Red-Dextranstaining of the metastases. FIG. 7C shows the autoradiogram of¹²⁵I-labeled trastuzumab, and indicates the fold increase in antibodyrelative to the surrounding normal brain tissue. FIG. 7D shows thetissue to blood ratio of ¹²⁵I-labeled trastuzumab in various tissues.Here, trastuzumab is distributed in organs with a ratio tissue to bloodof about 0.2 to 0.3 for lungs and spleen, and distribution in the heartis fairly high relative to the liver. The distribution in normal braintissue and brain metastases is comparatively low (see the inset in FIG.7D). The ratio of 0.02 for brain/blood is very low and only marginallysuperior to that found in the vascular space (i.e., the amount oftrastuzumab found in the brain vasculature, which is between about 0.01and 0.02). Very little uptake is observed in brain metastases eventhough these metastases demonstrate significant leakiness (see FIG. 7B).

FIGS. 8A-8F show the distribution of ¹²⁵I-labeled p97-trastuzumab in themouse brain and other tissues at two hours post-intravenousadministration. FIG. 8A shows brain metastases of heterogenous sizewithin the regions outlined in red, and FIG. 8B shows Texas Red-Dextranstaining of the metastases. FIG. 8C shows an autoradiogram of¹²⁵I-labeled p97-trastuzumab conjugates, and the left of FIG. 8Cindicates the amount (ng/g) of conjugate found in each metastases. Theleft of FIG. 8B shows the fold increase of p97-trastuzumab conjugatefound in each metastases, relative to the brain distant to tumor (BDT)region shown in FIG. 8A. FIG. 8D shows the tissue/blood ratio ofp97-trastuzumab conjugate for a variety of tissues. Here, distributionto the heart is significantly less than distribution to other tissues(e.g., about 10× less than lung, liver, and spleen). FIG. 8E shows theratio of p97-trastuzumab conjugate in normal brain/blood and brainmetastases/blood. The ratio for normal brain/blood is about 0.06(compared to 0.04 for ¹²⁵I-labeled p97 alone, data not shown), and theratio for brain metastases/blood is about 0.14 (compared to 0.06 for¹²⁵I-labeled p97 alone, data not shown). FIG. 8F summarizes theconcentration of ¹²⁵I-labeled p97-trastuzumab conjugate found inindividual brain metastases, with concentrations ranging from about25-175 ng/g tissue.

FIGS. 9A-9F show the distribution of ¹²⁵I-labeled p97-trastuzumab in themouse brain and other tissues at eight hours post-intravenousadministration. FIG. 9A shows brain metastases of heterogenous sizewithin the regions outlined in red, and FIG. 9B shows Texas Red-Dextranstaining of the metastases. FIG. 9C shows an autoradiogram of¹²⁵I-labeled p97-trastuzumab conjugate, and the left of FIG. 9Cindicates the amount (ng/g) of conjugate found in each metastases. Theleft of FIG. 9B shows the fold increase of p97-trastuzumab conjugatefound in each metastases, relative to the brain distant to tumor (BDT)regions shown in FIG. 9A. FIG. 9D shows the tissue/blood ratio ofp97-trastuzumab conjugate for a variety of tissues. Here, distributionof p97-trastuzumab to the heart is significantly less than distributionto other tissues. FIG. 9E shows the ratio of p97-trastuzumab conjugatein normal brain/blood and brain metastases/blood, where the ratio innormal brain/blood is about 0.04 (compared to 0.06 for the two-hour timepoint, see FIG. 8E), and the ratio in brain metastases/blood is about0.44 (compared to 0.14 for the two hour time point, see FIG. 8E). FIG.9F summarizes the concentration of ¹²⁵I-labeled p97-trastuzumabconjugate found in individual brain metastases, with concentrationsranging from about 25-125 ng/g tissue.

FIGS. 10A-10E summarize the data from the two and eight hour time pointsfollowing intravenous administration of ¹²⁵I-labeled p97-trastuzumabconjugate. FIG. 10A shows the tissue/blood ratio of p97-trastuzumabconjugate for a variety of tissues. The ratios do not vary much betweenthe two and eight hour time points; however, the ratio (levels) ofconjugate in heart tissue are significantly lower than other tissues(e.g., about 10× lower that lung and liver tissues). In contrast, thedistribution of trastuzumab alone in heart tissue was similar to liver(see FIG. 7D). FIG. 10B shows that the levels of conjugate in normalbrain tissue are marginally lower at the eight hour time point (relativeto the two hour time point), and the levels of conjugate in brainmetastases are significantly higher at the that same time point. FIG.10C shows the measured K_(in) values for the p97-trastuzumab conjugatein normal brain tissue (1.1×10⁻⁴ mL/sec/g) and brain metastases(4.9×10⁻⁴ mL/sec/g). Compared to the K_(in) values for trastuzumab (seeFIG. 6F), the p97-trastuzumab conjugate is transported into brain about1000 times more rapidly than trastuzumab alone. FIG. 10D shows thepercentage of injected dose in brain tissue at 2 and 8 hours, and FIG.10E summarizes the concentration of ¹²⁵I-labeled p97-trastuzumabconjugate in individual brain metastases at two and eight hourspost-administration.

The pharmacokinetic profile of p97-trastuzumab conjugates was alsocalculated based on the data from the two and eight hour time points.These data are shown in Table 9 below.

TABLE 9 Parameter Value Unit K_(e) 0.297 hr⁻¹ V_(d) 10.76 mL t_(1/2)2.32 hr Cl 3.202 mL/hr AUC_(0-∞) 2.523 μCi × hr/mL Dose 8.08 μCi/mL F 1[Unit-less]

Overall, these data strongly suggest that therapeutically effectiveconcentrations of p97-trastuzumab conjugate can be achieved in braintissue metastases, even by systemic (e.g., intravenous) administrationof such conjugates. These data also suggest that p97 and trastuzumabwork synergistically together to selectively target p97-trastuzumabconjugates to brain metastases relative to normal brain tissue, and at asignificantly greater rate (˜1000 fold) than trastuzumab alone.Conjugation to p97 thus not only increases transport of trastuzumabacross the blood-brain barrier, but also the blood-tumor barrier.Further, because of the reduced distribution to heart tissues relativeto other tissues, these data suggest that conjugation to p97 mightreduce the cardiotoxic effects of antibodies such as trastuzumab.

Example 5 Production of p97-Cetixumab Conjugates and Assays for In VitroCytotoxicity

In vitro anticancer efficacy assays are performed in two human cancercell lines, A-431 and HT-29, to evaluate the relative efficacies as theIC₅₀ of cetuximab and p97-cetuximab conjugates, relative to p97 aloneand phosphate buffer saline (PBS) as vehicle controls. The A-431 cellline is a EGFR-expressing human epidermoid carcinoma and the HT-29 cellline is a human colorectal adenocarcinomas.

As noted above, p97 (melanotransferrin) is a monomeric sialoglycoproteinbelonging of the iron binding family of proteins that includetransferrin, lactoferrin, and ovatransferrin. It was identifiedoriginally as a 97 kD, GPI linked, membrane bound protein on the surfaceof melanoma cells and was designated as melanoma-associated antigen p97.A soluble form (82 kD) missing the GPI anchor has been produced usingrecombinant techniques. Cetuximab (Erbitux®) is a human IgG₁ monoclonalantibody drug approved for use to treat certain human cancers by actingon the extracellular domain of EGFR and its mechanism of action isrelated to blocking EGFR activation by interfering with ligand binding.

p97-cetuximab conjugates were prepared by covalently linking soluble p97to cetuximab via a thioether linker. FIG. 11A shows an HPLC profile ofthe crude reaction mixture after 24 hours at room temperature.

The reaction product was then analyzed by size exclusion HPLC todetermine concentration and by SDS-PAGE to determine molecular weight.An aliquot of the concentrated, sterilized p97-cetixumab conjugate wasdiluted 10 times with 1×PBS, and an aliquot was injected into the HPLCsize exclusion column. As shown in FIG. 11B, the elution profile showeda major peak (96%) with a Rt of 8.875 minutes, earlier than that ofcetuximab (9.70 min) or p97 (10.03 min). The area of the peak at 220 nm(1856.9) was used together with p97 and cetuximab standard curves todetermine the concentration of the product at 4.0 mg/ml.

For SDS-PAGE analysis, samples of purified proteins or the reactionmixture (1 μg each) were analyzed on a 4%-12% bis-tris gel with 1×MESSDS running buffer under non-reducing conditions (Invitrogen NuPAGE®Novex® 1-12% Bis-Tris midi gel). The gel was run at a constant 125V for150 minutes and stained with SimplyBlue™ SafeStain (Invitrogen). Asshown in FIG. 12, the molecular weight of the conjugate was estimated tobe about 230 KDa based on the comparison to the protein ladder molecularweight standards; this estimate is consistent with a 1:1 p97-cetuximabratio.

For in vitro activity assays, A-431 cells are first propagated inATCC-formulated Dulbecco's Modified Eagle's Medium including 10% FBS,and HT-29 cells are propagated in ATCC-formulated McCoy's 5a MediumModified including 10% FBS. To ensure reliable IC₅₀ assay results,optimal culture conditions are verified based on cell morphology bymicroscopy, proliferation doubling time monitored by trypan blueexclusion assay, and cell split ratio data prior to treatment with testagents.

IC₅₀ assays are performed in flat bottom 96-well plates in triplicatefor each test agent concentration point and all controls at two fetalbovine serum (FBS) concentrations of 1% and 10% during the treatmentincubation period of 3 days. The test agents and controls areadministered to each of the cell line cultures at eight concentrationscovering a 1000-fold dose range based on the IC₅₀ of cetuximab inrelated human tumor cell lines. Specifically, a total of eight testagent concentrations over a semi-logarithmic concentration scale areevaluated for each of the two test agents, cetuximab and p97-cetuximab,and the control (p97) and vehicle (PBS) in PBS at the concentrationsshown in Table 10 below.

TABLE 10 Test Articles and Controls Test Concentrations (μg/mL)Cetuximab (positive control) 0.1, 0.3, 1.0, 3.0, 10, 30, 100 and 300P97-cetuximab 0.1, 0.3, 1.0, 3.0, 10, 30, 100 and 300 MTf 0.1, 0.3, 1.0,3.0, 10, 30, 100 and 300 PBS (vehicle control) 10% spike volume toincubation medium

Following 72 hours incubation of the test agents in each of the two cellcultures, MTT cytotoxicity assay are performed for evaluation of cellapoptosis according to BRI SOP: SOP-TM-GEN-029 (MTT Assay). IC₅₀ valuesand drug response curves are then generated with a SpectraMax™ M2 platereader operated by SpectraMax™ software (Molecular Devices) andSigmaPlot™ V5.0.

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent application, foreign patents, foreign patentapplication and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet areincorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary to employ concepts of thevarious patents, application and publications to provide yet furtherembodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

1-62. (canceled)
 63. A conjugate comprising a p97 polypeptide covalentlyor operatively linked to an antibody or antigen-binding fragment thereofthat specifically binds human Her1/EGFR (epidermal growth factorreceptor), wherein the p97 polypeptide is a soluble p97 polypeptide thathas a deletion of all or a portion of the hydrophobic domain as definedby residues 710-738 of SEQ ID NO: 1, and where the p97 polypeptide iseffective for transporting the anti-Her1/EGFR antibody orantigen-binding fragment thereof across the blood brain barrier.
 64. Theconjugate of claim 63, wherein the p97 polypeptide consists of residues20-711 of SEQ ID NO: 1 or differs from residues 20-711 of SEQ ID NO: 1by substitution, deletion, addition, and/or insertion of five aminoacids or fewer.
 65. The conjugate of claim 63, wherein the p97polypeptide and the antibody or antigen-binding fragment are covalentlylinked as a fusion polypeptide.
 66. The conjugate of claim 63, whereinthe p97 polypeptide is covalently linked to the antibody orantigen-binding fragment with a linker.
 67. The conjugate of claim 63,wherein the p97 polypeptide is (a) covalently linked to the antibody orantigen-binding fragment with a polymeric cross-linker, (b) covalentlylinked to the antibody or antigen-binding fragment via a nanoparticle,or (c) operatively linked to the antibody or antigen-binding fragmentthereof via a liposome.
 68. The conjugate of claim 63, wherein the p97polypeptide is covalently linked to the antibody or antigen-bindingfragment with a polymeric cross-linker comprising a thioether linkage.69. The conjugate of claim 63, wherein the p97 polypeptide is covalentlylinked to the antibody or antigen-binding fragment with a polymericcross-linker comprising polyethylene glycol.
 70. The conjugate of claim63, wherein the antibody or antigen-binding fragment thereof is specificfor human Her1/EGFR having a sequence set forth in SEQ ID NO:
 15. 71.The conjugate of claim 70, wherein the antibody is cetuximab or anantigen-binding fragment thereof.
 72. The conjugate of claim 63, wherethe conjugate is covalently linked to a cytotoxic agent.
 73. Theconjugate of claim 72, where the conjugate is covalently linked to thecytotoxic agent via the antibody.
 74. The conjugate of claim 73, wherethe cytotoxic agent is covalently linked to the antibody with a linker.75. The conjugate of claim 72, where the conjugate is covalently linkedto the cytotoxic agent via the p97 polypeptide.
 76. The conjugate ofclaim 75, where the cytotoxic agent is covalently linked to the p97polypeptide with a linker.
 77. A pharmaceutical composition comprising aconjugate of claim 63 or 71 and a pharmaceutically acceptable carrier,diluent, or excipient.
 78. A method for the treatment of a subject witha Her1/EGFR-expressing cancer comprising administering to the subject apharmaceutical composition of claim
 77. 79. The method of claim 78,where the cancer is a metastatic colorectal cancer or a head and neckcancer.
 80. The method of claim 79, where the cancer is anEGFR-expressing metastatic colorectal cancer.
 81. The method of claim80, where the colorectal cancer is KRAS wild-type.
 82. The method ofclaim 78, where the conjugate is administered after failure of bothirinotecan- and oxiplatin-based regimens.
 83. The method of claim 78,where the subject is intolerant to irinotecan-based regimens or isrefractory to irinotecan-based chemotherapy.
 84. The method of claim 79,where the cancer is a locally or regionally advanced squamous cellcarcinoma of the head and neck, a recurrent locoregional disease ormetastatic squamous cell carcinoma of the head and neck, or a recurrentor metastatic squamous cell carcinoma of the head and neck progressingafter platinum-based therapy.
 85. The method of claim 84, where theconjugate is administered in combination with radiation therapy,platinum-based therapy, or platinum-based therapy with 5-FU.